Production of spider silk protein in corn

ABSTRACT

Methods for the production of synthetic spider silk-like proteins in corn endosperm, plant leaf or plant shoot tissue are provided. The present invention provides further methods for the identification of synthetic spider silk-like proteins in corn endosperm, plant leaf or plant shoot tissue.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application in a non-provisional patent application of and claimspriority to U.S. Provisional Patent Application No. 61/348,997, filed onMay 27, 2010, and is a continuation of U.S. application Ser. No.13/117,643, filed on May 27, 2011, both of which are herein incorporatedby reference in their entirety.

ACKNOWLEDGEMENT OF FEDERAL RESEARCH SUPPORT

This invention was made, at least in part, with government support underNSF DBI#0501862 awarded by the National Science Foundation and NIH#EB000490 awarded by the National Institute of Health. Accordingly, theUnited States government has certain rights in this invention.

FIELD

The present disclosure relates to the field of molecular biology andplant genetics. More specifically, disclosed is one or more methods toproduce spider silk and synthetic spider silk-like proteins in planttissue such as plant endosperm tissue or shoot tissue (including shootmeristem, other non-photosynthetic tissue and leaf tissue). Alsodisclosed are methods to identify the presence of spider silk andsynthetic spider silk-like proteins expressed in plant endosperm tissueor shoot tissue.

SUBMISSION OF SEQUENCE LISTING

The Sequence Listing associated with this application is filed inelectronic format via EFS-Web and hereby incorporated by reference intothe specification in its entirety.

BACKGROUND

Increasing demands for materials and fabrics that are both lightweightand flexible without compromising strength and durability has created aneed for new fibers possessing higher tolerances for such properties aselasticity, denier, tensile strength and modulus. The search for abetter fiber has led to the investigation of fibers produced in nature,some of which possess remarkable qualities. One of those fibers isderived from spider or insect silk, which includes a group of externallyspun fibrous protein secretions.

Silks are produced by over 30,000 species of spiders and by many otherinsects, particularly in the order Lepidoptera. Few of these silks havebeen studied in detail. The cocoon silk of the domesticated silkwormBombyx mori and the dragline silk of the orb-weaving spider Nephilaclavipes are among the best characterized. Although the structuralproteins from the cocoon silk and the dragline silk are quite differentfrom each other in their primary amino acid sequences, they shareremarkable similarities in many aspects. They are extremely glycine andalanine-rich proteins. Fibroin, a structural protein of the cocoon silk,contains 42.9% glycine and 30% alanine. Spidroin 1, a major component ofthe dragline silk, contains 37.1% glycine and 21.1% alanine. They arealso highly repetitive proteins. The conserved crystalline domains inthe heavy chain of the Fibroin and a stretch of polyalanine in Spidroin1 are repeated numerous times throughout entire molecules. Thesecrystalline domains are surrounded by larger non-repetitive amorphousdomains in every 1 to 2 kilobases in the heavy chain of Fibroin, and byshorter repeated GXG amorphous domains in tandem in Spidroin 1. They arealso shear sensitive due to their high copy number of the crystallinedomains. During fiber spinning, the crystalline repeats are able to formanti-parallel-pleated sheets, so that silk protein is turned intosemi-crystalline fiber with amorphous flexible chains reinforced bystrong and stiff crystals.

Spider dragline silk has a tensile strength of over 200 ksi with anelasticity of nearly 35%, which makes it more difficult to break thaneither KEVLAR™ fibers or steel. When spun into fibers, spider silk mayhave application in the bulk clothing industries as well as beingapplicable for certain kinds of high strength uses such as rope,surgical sutures, flexible tie downs for certain electrical componentsand even as a biomaterial for implantation (e.g., artificial ligamentsor aortic banding). Additionally these fibers may be mixed with variousplastics and/or resins to prepare a fiber-reinforced plastic and/orresin product.

Traditional silk production from silkworm involves growing mulberryleaves, raising silkworms, harvesting cocoons, and processing of silkfibers. It is labor intensive and time consuming and thereforeprohibitively expensive. The natural defects of the silkworm silk, suchas the tendency to wrinkle and the irregularity of fiber diameterfurther limits its application. Similarly, the mass production of thedragline silk from spiders is not plausible because only small amountsare available from each spider. Furthermore, multiple forms of spidersilks are produced simultaneously by any given spider. The resultingmixture has less application than a single isolated silk because thedifferent spider silk proteins have different properties and are noteasily separated. Thus, the prospect of producing commercial quantitiesof spider silk from a natural source is not a practical one and thereremains a need for an alternate mode of production.

By using molecular recombination techniques, one can introduce foreigngenes or artificially synthesized DNA fragments into different hostorganisms for the purpose of expressing desired protein products incommercially useful quantities. Such methods usually involve joiningappropriate fragments of DNA to a vector molecule, which is thenintroduced into a recipient organism by transformation. Transformantsare selected using a selectable marker on the vector, or by a genetic orbiochemical screen to identify the cloned fragment.

While the techniques of foreign gene expression in the host cell arewell known and widely practiced, the synthesis of foreign polypeptidescontaining high numbers of repeating units poses unique problems. Genesencoding proteins of this type are prone to genetic instability due tothe repeating sequences, which result in truncated product instead ofthe full size protein.

The recent advances in cDNA sequencing of cocoon silk and dragline silkhave permitted the synthesis of artificial genes for spider silk-likeproteins with sequence and structural similarity to the native proteins.These artificial genes mimicked sequence arrays of natural cocoon silkfrom B. mori and dragline silk from N. clavipes, and had been introducedinto microorganisms such as Escherichia coli, Pichia pastoris, andSaccharomyces cerevisiae. Synthetic spider silk proteins have beenproduced in these microorganisms through fermentation.

Many recombinant proteins have been produced in transgenic plants. Plantgenetic engineering combines modern molecular recombination technologyand agricultural crop production. However there are strikingcompositional and structural differences between silks and spidersilk-like proteins and native plant proteins. For example, spidersilk-like proteins are very glycine and alanine-rich, highly repetitive,and semi-crystalline in structure. These are characteristics not foundin most plant proteins. Thus, introduction and expression of spidersilk-like proteins genes in plant cells may pose a number ofdifficulties. For example, the repetitive sequence of spider silk-likeprotein genes may be a target for DNA deletion and rearrangement inplant cells.

Alternatively, translation of glycine and alanine-rich spider silk-likeproteins might prematurely exhaust glycine and alanine and tRNA pools inplant cells. Finally, accumulation of semi-crystalline spider silk-likeproteins may be recognized and degraded by the housekeeping mechanismsin the plant.

The methods known in the art for the expression of spider silk andspider silk-like proteins are useful for production in microbialsystems. However, they are not applicable to the production of silk orspider silk-like proteins in plants. The use of a plant platform, suchas maize cells for the production of silk and silk-like proteins, hasseveral advantages over a microbial platform. For example, as arenewable resource, a plant platform requires far less energy andmaterial consumption than microbial methods. Similarly, a plant platformrepresents a far greater available biomass for protein production than amicrobial system.

There are several advantages of expressing spider silk proteins inplants over existing technologies. Corn endosperm, in particular, storeshigh concentrations of proteins in storage bodies, and targeting andprocessing can be directed by plant specific sequences.

The problem to be solved therefore is to provide a method to producesynthetic spider silk in the endosperm, leaf or shoot tissue of plantsand to easily identify when synthetic spider silk proteins have beenexpressed in the plant endosperm, leaf or shoot tissue.

The foregoing examples of the related art and limitations relatedtherewith are intended to be illustrative and not exclusive. Otherlimitations of the related art will become apparent to those of skill inthe art upon a reading of the specification.

SUMMARY

It is to be understood that the present invention includes a variety ofdifferent versions or embodiments, and this Summary is not meant to belimiting or all-inclusive. This Summary provides some generaldescriptions of some of the embodiments, but may also include some morespecific descriptions of other embodiments.

An embodiment of the present invention provides DNA constructs for theexpression of spider silk proteins in plant endosperm. Such DNAconstructs may be represented as PeUrr-SS-FP-X or PeUrr-FP-SS-X whereinPeUrr is a plant endosperm upstream regulatory region (URR, whichincludes upstream regulatory sequence, promoter region, transcriptionalstart site and a translation start codon), SS is a synthetic spider silkprotein coding sequence, FP is a fluorescent protein coding sequence andX is downstream regulatory region (DRR, including a translational stopsequence, transcription terminator sequence and downstream regulatoryregion). Further, the DNA construct is stably integrated into a plantDNA genome under conditions suitable for the expression of the DNAconstruct in a plant endosperm, where the DNA construct expresses aprotein in the plant endosperm. The expressed protein is a spider silkprotein with a fluorescent marker indicating successful integration andexpression.

An embodiment of the present invention provides DNA constructs for theexpression of spider silk proteins in corn plant endosperm. Such DNAconstructs are represented as CeUrr-SS-FP-X or CeUrr-FP-SS-X whereinCeUrr is a corn plant endosperm upstream regulatory region, SS is asynthetic spider silk protein coding sequence, FP is a fluorescentprotein coding sequence and X is a downstream regulatory region.Further, the DNA construct is stably integrated into a plant DNA genomeunder conditions suitable for the expression of the DNA construct in acorn plant endosperm, where the DNA construct expresses a protein in thecorn plant endosperm. The expressed protein is a spider silk proteinwith a fluorescent marker indicating successful integration andexpression.

An embodiment of the present invention provides DNA constructs for theexpression of spider silk proteins in plant shoot tissue (which includesleaf, meristematic and other non-photosynthetic tissue). Such DNAconstructs may be represented as PsUrr-SS-FP-X or PsUrr-FP-SS-X whereinPsUrr is a plant shoot tissue upstream regulatory region, SS is asynthetic spider silk protein coding sequence, FP is a fluorescentprotein coding sequence and X is a downstream regulatory region.Further, the DNA construct is stably integrated into a plant DNA genomeunder conditions suitable for the expression of the DNA construct in aplant shoot, where the DNA construct expresses a protein in the plantshoot. The expressed protein is a spider silk protein with a fluorescentmarker indicating successful integration and expression.

In an another embodiment of the present invention a DNA construct isprovided which comprises a nucleic acid having the sequence comprising aplant endosperm tissue promoter selected from the group comprising SEQID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6 and SEQ IDNO:7 where the plant endosperm tissue promoter is operably linked to asynthetic spider silk protein coding sequence selected from the groupcomprising SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ IDNO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ IDNO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ IDNO:39, and SEQ ID NO:40.

An embodiment of the present invention provides DNA construct having anucleic acid having the sequence comprising a corn plant endospermtissue promoter selected from the group comprising SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6 and SEQ ID NO:7 and wherethe corn plant endosperm tissue promoter is operably linked to asynthetic spider silk protein coding sequence selected from the groupcomprising SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ IDNO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ IDNO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ IDNO:39, and SEQ ID NO:40.

In an another embodiment of the present invention a DNA construct isprovided which comprises a nucleic acid having the sequence comprising aplant shoot-tissue promoter selected from the group comprising SEQ IDNO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:61, SEQ ID NO:62, and SEQID NO:68 where the shoot tissue promoter is operably linked to asynthetic spider silk protein coding sequence selected from the groupcomprising SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ IDNO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ IDNO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ IDNO:39, and SEQ ID NO:40.

In an another embodiment of the present invention a DNA construct isprovided which further comprises a synthetic spider silk protein codingsequence operably linked to a transcription terminator sequence.

In an another embodiment of the present invention a DNA construct isprovided which further comprises a sortable marker operably linked tothe 5′ end of said synthetic spider silk protein coding sequence wherethe sortable marker is selected from the group comprising SEQ ID NO:12,SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17,SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48,SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, and SEQ IDNO:53.

In an another embodiment of the present invention a DNA construct isprovided which further comprises a sortable marker operably linked tothe 3′ end of said synthetic spider silk protein coding sequence, wherethe sortable marker is selected from the group comprising SEQ ID NO:12,SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17,SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48,SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, and SEQ IDNO:53.

In an another embodiment of the present invention a transgenic plant isprovided having a DNA construct stably integrated into the DNA constructgenome under conditions suitable for the expression of the DNA constructin a plant endosperm, where the DNA construct expresses a protein in theplant endosperm. The expressed protein is a spider silk protein.

In an another embodiment of the present invention a method is providedfor producing synthetic spider silk proteins in the tissue of plantendosperm which comprises growing a transgenic plant having a DNAconstruct stably integrated into the DNA genome under conditionssuitable for the expression of the DNA construct in a plant endosperm.The DNA construct expresses a protein in the plant endosperm, whereinthe expressed protein is a spider silk protein.

In an another embodiment of the present invention a transgenic cornplant is provided having a DNA construct stably integrated into the DNAgenome under conditions suitable for the expression of the DNA constructin a corn plant endosperm, where the DNA construct expresses a proteinin the plant endosperm. The expressed protein is a spider silk protein.

In an another embodiment of the present invention a method for producingsynthetic spider silk proteins in the tissue of corn plant endosperm isprovided which comprises growing a transgenic plant having a DNAconstruct stably integrated into the DNA genome under conditionssuitable for the expression of the DNA construct in a corn plantendosperm. The DNA construct expresses a protein in the corn plantendosperm, wherein the expressed protein is a spider silk protein.

In an another embodiment of the present invention a transgenic plant isprovided having a DNA construct stably integrated into the DNA constructgenome under conditions suitable for the expression of the DNA constructin a plant shoot, where the DNA construct expresses a protein in theplant shoot. The expressed protein is a spider silk protein.

In an another embodiment of the present invention to provide a methodfor producing synthetic spider silk proteins in the plant shoot whichcomprises growing a transgenic plant having a DNA construct stablyintegrated into the DNA genome under conditions suitable for theexpression of the DNA construct in a plant shoot. The DNA constructexpresses a protein in the plant shoot, wherein the expressed protein isa spider silk protein.

Various components are referred to herein as “operably linked”, “linked”or “operably associated.” As used herein, “operably linked”, “linked” or“operably associated” refers to nucleic acid sequences on a singlenucleic acid fragment so that the function of one is affected by theother. For example, a promoter is operably linked with a coding sequencewhen it is capable of affecting the expression of that coding sequence.

As used herein, “at least one,” “one or more,” and “and/or” areopen-ended expressions that are both conjunctive and disjunctive inoperation. For example, each of the expressions “at least one of A, Band C,” “at least one of A, B, or C,” “one or more of A, B, and C,” “oneor more of A, B, or C” and “A, B, and/or C” means A alone, B alone, Calone, A and B together, A and C together, B and C together, or A, B andC together.

As used herein, “sometime” means at some indefinite or indeterminatepoint of time. So for example, as used herein, “sometime after” meansfollowing, whether immediately following or at some indefinite orindeterminate point of time following the prior act.

Various embodiments of the present invention are set forth in theDetailed Description as provided herein and as embodied by the claims.It should be understood, however, that this Summary does not contain allof the aspects and embodiments of the present invention, is not meant tobe limiting or restrictive in any manner, and that the invention(s) asdisclosed herein is/are understood by those of ordinary skill in the artto encompass obvious improvements and modifications thereto.

Additional advantages of the present invention will become readilyapparent from the following discussion, particularly when taken togetherwith the accompanying drawings and sequence listings.

SEQUENCE LISTINGS

SEQ ID NO:1 discloses the leader nucleic acid sequence of one embodimentof the present invention.

SEQ ID NO:2 discloses the nucleic sequence for the Zein gene (floury2)(Genbank Accession Number MZEZFL2).

SEQ ID NO:3 discloses the amino acid sequence for the Zein gene(floury2) (Genbank Accession Number AAA76580).

SEQ ID NO:4 discloses the nucleic acid sequence which encodes theregulatory region of the endosperm tissue promoter of one embodiment ofthe present invention.

SEQ ID NO:5 discloses the nucleic acid sequence which encodes theregulatory region of the transcription terminator of one embodiment ofthe present invention.

SEQ ID NO:6 discloses the nucleic acid sequence which encodes theTriticum aestivum endosperm regulatory region containing the promoterregion and 5′ UTR of the (SbeIIa) gene (Genbank Accession NumberAY357072).

SEQ ID NO:7 discloses the nucleic acid sequence of nucleic acid sequencewhich encodes the regulatory region of the Opaque 2 gene endospermtissue promoter (Genbank Accession Number: FJ935743).

SEQ ID NO:8 discloses the nucleic acid sequence for the forward primer 1for the amplification of the promoter and the partial gene sequence ofSEQ ID NO:1.

SEQ ID NO:9 discloses the nucleic acid sequence for the reverse primer 2for the amplification of the promoter and the partial gene sequence ofSEQ ID NO:1 of the present invention.

SEQ ID NO:10 discloses the nucleic acid sequence for forward primer forthe amplification of the partial gene sequence of SEQ ID NO:1 and the 3′UTR which includes the transcription terminator.

SEQ ID NO:11 discloses the reverse primer for the amplification of thepromoter and gene sequence of SEQ ID NO:1.

SEQ ID NO:12 discloses the nucleic acid sequence of the Red FluorescentProtein (mRFP).

SEQ ID NO:13 discloses the protein sequence of the Red FluorescentProtein (mRFP).

SEQ ID NO:14 discloses the nucleic acid sequence of the Cyan FluorescentProtein (CFP) (Genbank Accession Number: AY646072).

SEQ ID NO:15 discloses the protein sequence of the Cyan FluorescentProtein (CFP) (Genbank Accession Number: AAU06851).

SEQ ID NO:16 discloses the nucleic acid sequence of the GreenFluorescent Protein pCmGFP (GFP) (Genbank Accession Number: FJ172221).

SEQ ID NO:17 discloses the protein sequence of the Green FluorescentProtein pCmGFP (GFP) (Genbank Accession Number: ACJ06700).

SEQ ID NO:18 discloses the nucleic acid sequence of the YellowFluorescent Protein (YFP) (Genbank Accession Number: GQ221700).

SEQ ID NO:19 discloses the protein sequence of the Yellow FluorescentProtein (YFP) (Genbank Accession Number: GQ221700).

SEQ ID NO:20 discloses the synthetic spider silk protein sequence E₄S₄of a Nephila clavipes MaSp 2 construct.

SEQ ID NO:21 discloses the synthetic spider silk protein sequence E₄S₈of a Nephila clavipes MaSp 2 construct.

SEQ ID NO:22 discloses the synthetic spider silk protein sequence E₄S₁₆of a Nephila clavipes MaSp 2 construct.

SEQ ID NO:23 discloses the synthetic spider silk protein sequence E₁₆S₈of a Nephila clavipes MaSp 2 construct.

SEQ ID NO:24 discloses the synthetic spider silk protein sequence E₁S₈of a Argiope sp. MaSp 2 construct.

SEQ ID NO:25 discloses the synthetic spider silk protein sequence E₂S₈of a Argiope sp. MaSp 2 construct.

SEQ ID NO:26 discloses the synthetic spider silk protein sequence E₃S₈of a Argiope sp. MaSp 2 construct.

SEQ ID NO:27 discloses the recombinant synthetic spider silk proteinsequences made up of the fusion protein sequence A1S8₂₀.

SEQ ID NO:28 discloses the recombinant synthetic spider silk proteinsequences made up of the fusion protein sequence A2S8₁₄.

SEQ ID NO:29 discloses the recombinant synthetic spider silk proteinsequences made up of the fusion protein sequence A4S8₈.

SEQ ID NO:30 discloses the recombinant synthetic spider silk proteinsequences made up of the fusion protein sequence A₄₀.

SEQ ID NO:31 discloses the recombinant synthetic spider silk proteinsequences made up of the fusion protein sequence Y1S8₂₀.

SEQ ID NO:32 discloses the recombinant synthetic spider silk proteinsequences made up of the fusion protein sequence Y2S8₁₄.

SEQ ID NO:33 discloses the recombinant synthetic spider silk proteinsequences made up of the fusion protein sequence Y4S8₈.

SEQ ID NO:34 discloses the recombinant synthetic spider silk proteinsequences made up of the fusion protein sequence Y₄₇.

SEQ ID NO:35 discloses the spider silk nucleic acid sequence made up ofthe sequence PXP.

SEQ ID NO:36 discloses the spider silk protein sequence made up of thesequence PXP.

SEQ ID NO:37 discloses the spider silk nucleic acid sequence made up ofthe sequence QQ.

SEQ ID NO:38 discloses the synthetic spider silk protein sequence madeup of the sequence QQ.

SEQ ID NO:39 discloses the synthetic spider silk nucleic acid sequencemade up of the full piriform sequence.

SEQ ID NO:40 discloses the synthetic spider silk protein sequence madeup of the full piriform sequence.

SEQ ID NO:41 discloses the nucleic acid sequence for forward primer forthe Red Fluorescent Protein (mRFP).

SEQ ID NO:42 discloses the nucleic acid sequence for reverse primer forthe Red Fluorescent Protein (mRFP).

SEQ ID NO:43 discloses the nucleic acid sequence for forward primer forCyan Fluorescent Protein (CFP) and Yellow Fluorescent Protein (YFP).

SEQ ID NO:44 discloses the nucleic acid sequence for reverse primer forthe Cyan Fluorescent Protein (CFP) and Yellow Fluorescent Protein (YFP).

SEQ ID NO:45 discloses the complete nucleic acid sequence containing thepromoter of one embodiment of the present invention, the Zein gene andthe transcription terminator.

SEQ ID NO:46 discloses the nucleic acid sequence of the maize specificTeal Fluorescent Protein (mTFP).

SEQ ID NO:47 discloses the protein sequence of the maize specific TealFluorescent Protein (mTFP).

SEQ ID NO:48 discloses the nucleic acid sequence of the maize specificBlue Fluorescent Protein (mBFP).

SEQ ID NO:49 discloses the protein sequence of the maize specific BlueFluorescent Protein (mBFP).

SEQ ID NO:50 discloses the nucleic acid sequence of the maize specificCherry Fluorescent Protein, mCherry (mChFP).

SEQ ID NO:51 discloses the protein sequence of the maize specific CherryFluorescent Protein, mCherry (mChFP).

SEQ ID NO:52 discloses the nucleic acid sequence of the maize specificCerulean Fluorescent Protein (mCeFP).

SEQ ID NO:53 discloses the protein sequence of the maize specificCerulean Fluorescent Protein (mCeFP).

SEQ ID NO:54 discloses the Nicotiana tabacum nucleic acid sequence whichencodes the regulatory region of the Dfr2 gene leaf tissue promoter, and5′ UTR (Genbank Accession Number FJ472649).

SEQ ID NO:55 discloses the Nicotiana tabacum nucleic acid sequence whichencodes the regulatory region of the Dfr2 gene leaf tissue promoter,(Genbank Accession Number FJ472649).

SEQ ID NO:56 discloses the Nicotiana plumbaginifolia nucleic acidsequence which encode the regulatory region of the Cab gene and leaftissue promoters. (Genbank Accession Number X12512).

SEQ ID NO:57 discloses the nucleic acid sequence for forward primer andlinker sequence for the maize specific blue (mBFP), cherry (mChFP) andteal (mTFP) Fluorescent Protein.

SEQ ID NO:58 discloses the nucleic acid sequence for reverse primer andlinker sequence for the maize specific blue (mBFP), cherry (mChFP) andteal (mTFP) Fluorescent Protein.

SEQ ID NO:59 discloses the nucleic acid sequence for forward primer andlinker sequence for the maize specific cerulean Fluorescent Protein(mCeFP).

SEQ ID NO:60 discloses the nucleic acid sequence for reverse primer andlinker sequences for the maize specific cerulean Fluorescent Protein(mCeFP).

SEQ ID NO:61 discloses the Zea mays nucleic acid sequence which encodesthe RAB2A gene shoot tissue regulatory region with the promoter and 3′UTR.

SEQ ID NO:62 discloses the Zea mays nucleic acid sequence which encodesthe RAB2A gene shoot tissue promoter.

SEQ ID NO:63 discloses the nucleic acid sequence for the primer 1 forthe amplification of the promoter and the partial gene sequence of SEQID NO:61 and SEQ ID NO:62.

SEQ ID NO:64 discloses the nucleic acid sequence for the reverse primer2 for the amplification of the promoter and the partial gene sequence ofSEQ ID NO:61 and SEQ ID NO:62 of the present invention.

SEQ ID NO:65 discloses the nucleic acid sequence for the forward primer3 for the amplification of the promoter and the partial gene sequence ofSEQ ID NO:61.

SEQ ID NO:66 discloses the nucleic acid sequence for the reverse primer4 for the amplification of the promoter and the partial gene sequence ofSEQ ID NO:61 and SEQ ID NO:62.

SEQ ID NO:67 discloses the nucleic acid sequence for the forward primer3 for the amplification of the promoter sequence of SEQ ID NO:62.

SEQ ID NO:68 discloses the Hordeum vulgare nucleic acid sequence whichencodes the myb2 gene shoot tissue promoter (Genbank Accession NumberX70876).

BRIEF DESCRIPTION OF THE DRAWINGS

To further clarify the above and other advantages and features of thepresent invention, a more particular description of the invention isrendered by reference to specific embodiments thereof which areillustrated in the appended drawings. It is appreciated that thesedrawings depict only typical embodiments of the invention and aretherefore not to be considered limiting of its scope. The invention isdescribed and explained with additional specificity and detail throughthe use of the accompanying drawings in which.

FIG. 1 is a map of a DNA construct, represented as PeUrr-SS-FP-X thatincludes (from 5′ to 3′), a plant endosperm tissue upstream regulatoryregion, a synthetic spider silk protein coding sequence, a fluorescentprotein coding sequence with linker sequences and a downstreamregulatory region including transcription terminator sequence.

FIG. 2 is a map of a DNA construct, represented as PeUrr-FP-SS-X thatincludes (from 5′ to 3′), a plant endosperm tissue upstream regulatoryregion, a fluorescent protein coding sequence with linker sequences, asynthetic spider silk protein coding sequence, and a downstreamregulatory region including transcription terminator sequence.

FIG. 3 is a map of a DNA construct, represented as CeUrr-FP-SS-X thatincludes (from 5′ to 3′), a corn endosperm tissue upstream regulatoryregion, a synthetic spider silk protein coding sequence, a fluorescentprotein coding sequence with linker sequences and a downstreamregulatory region including transcription terminator sequence.

FIG. 4 is a map of a DNA construct, represented as PsUrr-FP-SS-X thatincludes (from 5′ to 3′), a plant shoot tissue upstream regulatoryregion, a synthetic spider silk protein coding sequence, a fluorescentprotein coding sequence with linker sequences and a downstreamregulatory region including transcription terminator sequence.

The drawings are not necessarily to scale.

DETAILED DESCRIPTION

The present invention provides one or more methods for the expression ofspider silk and spider silk-like proteins in the endosperm or shoottissue of plants and in particular corn plants. The term “plant”includes reference to an immature or mature whole plant, including aplant from which seed, grain, anthers, or pistils have been removed. Aseed or embryo that will produce the plant is also considered to be theplant. The present invention further provides one or more methods forthe identification of spider silk and spider silk-like proteins in theendosperm or shoot tissue of a plant and in particular corn plants. Thespider silk and spider silk-like proteins of the present invention mayhave properties suitable for fabrics, or alternatively may be useful inmaterials.

In one or more embodiments of the present invention one or more DNAconstructs are provided for use in expression of synthetic spider silkin plant endosperm represented by PeUrr-SS-FP-X and variations thereof,by providing at least one plant endosperm tissue promoter operablylinked to at least one synthetic spider silk protein coding sequence.The synthetic spider silk protein is also operably linked to a sortablemarker which is operably linked to a transcription terminator sequence.Transgenic plants expressing the synthetic spider silk protein genes arethen generated. The preferred embodiment of the transgenic plants of thepresent invention is corn plants and corn plant endosperm promoterswherein the construct is represent by CeUrr-SS-FP-X and variationsthereof. However, the transgenic plants may include, but are not limitedto other plants, such as barley, rice, wheat, sorghum and millet.

In one or more embodiments of the present invention one or more DNAconstructs are provided for use in expression of synthetic spider silkin shoot tissue, represented by PsUrr-SS-FP-X and variations thereof, byproviding at least one plant shoot tissue promoter operably linked to atleast one synthetic spider silk protein coding sequence. The syntheticspider silk protein coding sequence is also operably linked to asortable marker which is operably linked to a transcription terminatorsequence. Transgenic plants expressing the synthetic spider silk proteingenes are then generated. The preferred embodiment of the transgenicplants of one embodiment of the present invention is but not limited to,corn plants. However, the transgenic plants may include, but are notlimited to other plants, such as barley, corn, tobacco, rice, wheat,sorghum and millet.

The present invention provides one or more recombinant constructs thatare suitable for the expression of spider silk proteins in plantendosperm such as corn and plant shoot tissue. As shown in FIG. 1, theconstruct of one embodiment of the present invention is generallyrepresented as PeUrr-SS-FP-X, 100, wherein PeUrr is the upstreamregulatory region including the plant endosperm tissue promoter, thetranscription start sequence, and the start codon (ATG) 102, SS is asynthetic spider silk protein coding sequence 104, FP, a sortablemarker, is a fluorescent protein region that includes a linker sequenceon the 5′ end of the fluorescent protein coding sequence and linkersequence on the 3′ end of the fluorescent protein coding 106 and X is adownstream regulatory region including the stop codon (TGA, TAG or TAA)and the transcription terminator sequence 108. Each of these fourcomponents is operably linked to the next, i.e., the plant endospermtissue upstream regulatory region is operably linked to the 5′ end ofthe synthetic spider silk sequence encoding the synthetic spider silkprotein, the synthetic spider silk protein coding sequence is operablylinked to the 5′ end of the fluorescent protein coding sequence and thefluorescent protein coding sequence is operably linked to 5′ end of thedownstream regulatory region. Synthetic spider silk protein may also beexpressed in variations of the construct of FIG. 1 including but notlimited to PeUrr-SS-X, PeUrr-SS-FP and PeUrr-SS.

As shown in FIG. 2, the construct of another embodiment of the presentinvention is generally represented as PeUrr-FP-SS-X, 200, wherein PeUrris the upstream regulatory region including the corn endosperm tissuepromoter, the transcription start sequence, and the start codon (ATG)202, FP, a sortable marker, is a fluorescent protein region thatincludes a linker sequence on the 5′ end of the fluorescent proteincoding sequence and linker sequence on the 3′ end of the fluorescentprotein coding, 204 SS is a synthetic spider silk protein codingsequence, 206 and X, the transcription terminator, is a downstreamregulatory region including the stop codon (TGA, TAG or TAA) and thetranscription terminator sequence 208. Each of these four components isoperably linked to the next, i.e., the plant endosperm upstreamregulatory region is operably linked to the 5′ end of the syntheticspider silk sequence encoding the synthetic spider silk protein, thesynthetic spider silk protein coding sequence is operably linked to the5′ end of the fluorescent protein coding sequence and the fluorescentprotein coding sequence is operably linked to 5′ end of the downstreamregulatory region. FIG. 2, including but not limited to PeUrr-SS-X,PeUrr-FP-SS and PeUrr-SS.

As shown in FIG. 3, the construct of another embodiment of the presentinvention is generally represented as CeUrr-SS-FP-X, 300, wherein CeUrris the upstream regulatory region including the corn endosperm tissuepromoter, the transcription start sequence, and the start codon (ATG)302, SS is a synthetic spider silk protein coding sequence 304, FP, asortable marker, is a fluorescent protein region that includes a linkersequence on the 5′ end of the fluorescent protein coding sequence andlinker sequence on the 3′ end of the fluorescent protein coding 306 andX, the transcription terminator, is a downstream regulatory regionincluding the stop codon (TGA, TAG or TAA) and the transcriptionterminator sequence 308. Each of these four components is operablylinked to the next, i.e., the corn endosperm tissue upstream regulatoryregion is operably linked to the 5′ end of the synthetic spider silksequence encoding the synthetic spider silk protein, the syntheticspider silk protein coding sequence is operably linked to the 5′ end ofthe fluorescent protein coding sequence and the fluorescent proteincoding sequence is operably linked to 5′ end of the downstreamregulatory region. Spider silk protein may also be expressed invariations of the construct of FIG. 3 including but not limited toCeUrr-SS-X, CeUrr-SS-FP, CeUrr-SS, CeUrr-FP-SS-X, and CeUrr-FP-SS.

As shown in FIG. 4, the construct of one embodiment of the presentinvention may be generally represented as PsUrr-SS-FP-X, 400, whereinPsUrr is the upstream regulatory region including the plant shoot tissuepromoter, the transcription start sequence, and the start codon (ATG)402, SS is a synthetic spider silk protein coding sequence 404, FP, asortable marker, is a fluorescent protein region that includes a linkersequence on the 5′ end of the fluorescent protein coding sequence andlinker sequence on the 3′ end of the fluorescent protein coding 406 andX is a downstream regulatory region including the stop codon (TGA, TAGor TAA) and the transcription terminator sequence 408. Each of thesefour components is operably linked to the next, i.e., the plant shoottissue upstream regulatory region is operably linked to the 5′ end ofthe synthetic spider silk sequence encoding the synthetic spider silkprotein, the synthetic spider silk protein coding sequence is operablylinked to the 5′ end of the fluorescent protein coding sequence and thefluorescent protein coding sequence is operably linked to 5′ end of thedownstream regulatory region. Synthetic spider silk protein may also beexpressed in variations of the construct of FIG. 4 including but notlimited to PsUrr-SS-X, PsUrr-SS-FP, PsUrr-SS, PsUrr-FP-SS-X, andPsUrr-FP-SS.

A variety of techniques are available and known to those skilled in theart for introduction of constructs into a plant cell host. Thesetechniques include transformation with DNA employing A. tumefaciens orA. rhizogenes as the transforming agent, electroporation, particleacceleration, etc. It is particularly preferred to use the binary typevectors of Ti and Ri plasmids of Agrobacterium spp. Ti-derived vectorstransform a wide variety of higher plants, including monocotyledonousand dicotyledonous plants, such as soybean, cotton, rape, tobacco, andrice. The use of T-DNA to transform plant cells has received extensivestudy and are known to those skilled in the art. For introduction intoplants, the chimeric genes of the invention can be inserted into binaryvectors as described in the examples.

Other transformation methods are available to those skilled in the art,such as direct uptake of foreign DNA constructs, techniques ofelectroporation or high-velocity ballistic bombardment with metalparticles coated with the nucleic acid constructs. Once transformed, thecells can be regenerated by those skilled in the art. Of particularrelevance are the methods to transform foreign genes into commerciallyimportant crops, such as rapeseed, sunflower, soybean, rice and corn.

Transgenic plant cells are then placed in an appropriate selectivemedium for selection of transgenic cells, which are then grown tocallus. (Please note that transgenic is used to indicate a plant, orphotosynthetic organism including algae, which has been geneticallymodified to contain the DNA constructs of the present invention.) Shootsare grown from callus and plantlets generated from the shoot by growingin rooting medium. The various constructs normally will be joined to amarker for selection in plant cells. Conveniently, the marker may beresistance to a biocide (particularly an antibiotic such as kanamycin,G418, bleomycin, hygromycin, chloramphenicol, herbicide, or the like).The particular marker used will allow for selection of transformed cellsas compared to cells lacking the DNA, which has been introduced. Aplasmid, vector or cassette which is a n extrachromosomal element isused to carry genes and usually in the form of circular double-strandedDNA molecules. Such elements may be autonomously replicating sequences,genome integrating sequences, phage or nucleotide sequences, linear orcircular, of a single- or double-stranded DNA or RNA, derived from anysource, in which a number of nucleotide sequences have been joined orrecombined into a unique construction which is capable of introducing apromoter fragment and DNA sequence for a selected gene product alongwith an appropriate 3′ untranslated sequence into a cell. Components ofDNA constructs including transcription cassettes of this invention maybe prepared from sequences which are native (endogenous) or foreign(exogenous) to the host. By “foreign” it is meant that the sequence isnot found in the wild-type host into which the construct is introduced.Heterologous constructs will contain at least one region, which is notnative to the gene from which the transcription initiation region isderived.

To confirm the presence of the transgenes in transgenic cells andplants, a polymerase chain reaction (PCR) amplification or Southern blotanalysis can be performed using methods known to those skilled in theart. A PCR refers to a scientific technique to amplify a single or a fewcopies of a piece of DNA across several orders of magnitude, generatingthousands to millions of copies of a particular DNA sequence. Thepresent invention provides one or more methods to generate geneconstructs including but not limited to the ttPCR method and theGateway® Multisite method. The ttPCR method uses triple template PCR togenerate the genomic sequence including 5′ UTR, genomic gene sequenceand 3′UTR with inserted fluorophor. A UTR refers to the untranslatedregion on either of two sections on either side of a coding sequence ona strand of mRNA. The ttPCR product is subsequently cloned using theGateway® recombination vectors. The Gateway® Multisite method usesthree-way or four-way multisite Gateway® cloning, which bypasses theneed to generate three PCR products and instead prepares three to fourGateway® clones, which are subsequently cloned together into the finalvector. The ttPCR method is efficient for small size genomic sequences<5kb in size. The Gateway® Multisite cloning method is used for final genesizes from 5-15 kb or larger.

Expression products of the transgenes can be detected in any of avariety of ways, depending upon the nature of the product, and includeWestern blot and enzyme assay. One particularly useful way to quantitateprotein expression and to detect replication in different plant tissuesis to use a reporter gene, such as GUS. Once transgenic plants have beenobtained, they may be grown to produce plant tissues or parts having thedesired phenotype. The plant tissue or plant parts, may be harvested,and/or the seed collected. The seed may serve as a source for growingadditional plants with tissues or parts having the desiredcharacteristics. Expression includes the process by which informationfrom a gene is used in the synthesis of a functional gene product, suchas the expression of spider silk proteins or synthetic spider silkproteins in the endosperm of maize. These products are often proteins,but in non-protein coding genes such as rRNA genes or tRNA genes, theproduct is a functional RNA. The process of gene expression is used byall known life, i.e., eukaryotes (including multicellular organisms),prokaryotes (bacteria and archaea), and viruses, to generate themacromolecular machinery for life. Several steps in the gene expressionprocess may be modulated, including the transcription, up-regulation,RNA splicing, translation, and post-translational modification of aprotein.

Generally, the DNA that is introduced into a plant is part of aconstruct. A construct is an artificially constructed segment of DNAthat may be introduced into a target plant tissue or plant cell. The DNAmay be a gene of interest, e.g., a coding sequence for a protein, or itmay be a sequence that is capable of regulating expression of a gene,such as an antisense sequence, a sense suppression sequence, or a miRNAsequence. The construct typically includes regulatory regions operablylinked to the 5′ side of the DNA of interest and/or to the 3′ side ofthe DNA of interest. Operably linked refers to the association ofnucleic acid sequences on a single nucleic acid fragment so that thefunction of one is affected by the other. For example, a promoter isoperably linked with a coding sequence when it is capable of affectingthe expression of that coding sequence (i.e., that the coding sequenceis under the transcriptional control of the promoter). Coding sequencescan be operably linked to regulatory sequences in sense or antisenseorientation. A cassette containing all of these elements is alsoreferred to herein as an expression cassette. The expression cassettesmay additionally contain 5′ leader sequences in the expression cassetteconstruct. (A leader sequence is a nucleic acid sequence containing apromoter as well as the upstream region of a gene.) The regulatoryregions (i.e., promoters, transcriptional regulatory regions, andtranslational termination regions) and/or the polynucleotide encoding asignal anchor may be native/analogous to the host cell or to each other.Alternatively, the regulatory regions and/or the polynucleotide encodinga signal anchor may be heterologous to the host cell or to each other.The expression cassette may additionally contain selectable markergenes. Targeting constructs are engineered DNA molecules that encodegenes and flanking sequences that enable the constructs to integrateinto the host genome at (targeted) locations. Publicly availablerestriction enzymes may be used for the development of the constructs ofthe present invention. Targeting constructs depend upon homologousrecombination to find their targets.

Other heterologous proteins encoded by the chimeric gene includepolypeptides that form immunologically active epitopes, and enzymes thatcatalyze conversion of intracellular metabolites, with the consequentbuild-up of selected metabolites in the cells.

The expression cassette or chimeric genes in the transforming vectortypically have a transcriptional termination region at the opposite endfrom the transcription initiation regulatory region. The transcriptionaltermination region may normally be associated with the transcriptionalinitiation region from a different gene. The transcriptional terminationregion may be selected, particularly for stability of the mRNA, toenhance expression. Illustrative transcriptional termination regionsinclude the NOS terminator from Agrobacterium Ti plasmid and the riceα-amylase terminator.

A promoter is a DNA region, which includes sequences sufficient to causetranscription of an associated (downstream) sequence. The promoter maybe regulated, i.e., not constitutively acting to cause transcription ofthe associated sequence. If inducible, there are sequences presenttherein which mediate regulation of expression so that the associatedsequence is transcribed only when an inducer molecule is present. Thepromoter may be any DNA sequence which shows transcriptional activity inthe chosen plant cells, plant parts, or plants. The promoter may beinducible or constitutive. It may be naturally-occurring, may becomposed of portions of various naturally-occurring promoters, or may bepartially or totally synthetic. Also, the location of the promoterrelative to the transcription start may be optimized. Many suitablepromoters for use in plants are well known in the art, as are nucleotidesequences, which enhance expression of an associated expressiblesequence.

A tissue-specific promoter of the DNA constructs of one or moreembodiments of the present invention is a regulated promoter that is notexpressed in all plant cells but only in one or more cell types inspecific organs (such as leaves or seeds), specific tissues (such asembryo or cotyledon), or specific cell types (such as leaf parenchyma orseed storage cells). This also includes promoters that are temporallyregulated, such as in early or late embryogenesis, during fruit ripeningin developing seeds or fruit, in fully differentiated leaf, or at theonset of senescence. While the tissue promoter in the regulatory regionupstream of the leader sequence of the Zein gene (SEQ ID NO:1) is oneexample of a promoter of a construct of one embodiment of the presentinvention, a number of promoters may be used in the practice of theconstructs of the present invention, including but not limited to theTriticum aestivum endosperm (SbeIIa) promoter (SEQ ID NO:6) (GenbankAccession Number AY357072), the Opaque 2 gene endosperm tissue promoter(SEQ ID NO:7) (Genbank Accession Number FJ935743), the tobacco Dfr2 geneleaf tissue promoters, (SEQ ID NO: 54) and (SEQ ID NO: 55) (GenbankAccession Number FJ472649), the tobacco Cab gene leaf tissue promoter(SEQ ID NO: 56) (Genbank Accession Number X12512), the Zea mays shoottissue RAB2A gene (SEQ ID NO:61), the Zea mays shoot tissue RAB2Apromoter (SEQ ID NO:62) and the Hordeum vulgare myb2 gene shoot tissuepromoter (SEQ ID NO:68). The promoters can be selected based on thedesired outcome. That is, the nucleic acids can be combined withconstitutive, tissue preferred, or other promoters for expression in thehost cell of interest. The promoter may be inducible or constitutive. Itmay be naturally-occurring, may be composed of portions of variousnaturally-occurring promoters, or may be partially or totally synthetic.Guidance for the design of promoters is commonly known in the art. Inaddition, the location of the promoter relative to the transcriptionstart may be optimized. Many suitable promoters for use in plants arewell known in the art, as are nucleotide sequences, which enhanceexpression of an associated expressible sequence.

Several tissue-specifically regulated genes and/or promoters have beenreported in plants. These include genes encoding the seed storageproteins (such as napin, cruciferin, beta-conglycinin, glycinin andphaseolin), zein or oil body proteins (such as oleosin), or genesinvolved in fatty acid biosynthesis (including acyl carrier protein,stearoyl-ACP desaturase, and fatty acid desaturases (fad 2-1)), andother genes expressed during embryo development (such as Bce4).Particularly useful for seed-specific expression is the pea vicilinpromoter. Other useful promoters for expression in mature leaves arethose that are switched on at the onset of senescence, such as the SAGpromoter from Arabidopsis.

The promoter may include, or be modified to include, one or moreenhancer elements. Preferably, the promoter will include a plurality ofenhancer elements. Promoters containing enhancer elements provide forhigher levels of transcription as compared to promoters that do notinclude them. Suitable enhancer elements for use in plants include thePClSV enhancer element and the CaMV 35S enhancer element.

Preparation of Spider Silk-Encoding Nucleic Acid Molecules, Spider SilkProteins, and Antibodies Thereto

Spider silk or silk-like proteins refer to natural silk proteins andtheir synthetic analogs having the following three criteria: (1) Aminoacid composition of the molecule is dominated by glycine and/or alanine;(2) Consensus crystalline domain is arrayed repeatedly throughout themolecule; (3) the molecule is shear sensitive and can be spun intosemicrystalline fiber. Spider silk proteins should also includemolecules which are the modified variants of the natural silk proteinsand their synthetic analogs defined above.

There are a variety of spider silks, which may be suitable forexpression in plants. Many of these are derived from the orb-weavingspiders such as those belonging to the genus Nephila. Silks from thesespiders may be divided into major ampullate, minor ampullate, andflagelliform silks, each having different physical properties. Those ofthe major ampullate are the most completely characterized and are oftenrefereed to as spider dragline silk. Natural spider dragline consists oftwo different proteins that are co-spun from the spider's majorampullate gland.

The present invention provides for various silk and synthetic silk-likeproteins in the constructs of the present invention for the expressionin the endosperm, leaf or shoot tissue of plants. Of particular interestare the synthetic silks which have as a repeating unit(GPGGYGPGQQ)₄GPGGPSGPGSAAAA (SEQ ID NO:20),(GPGGYGPGQQ)₄GPGGPSGPGSAAAAAAAA (SEQ ID NO:21)(GPGGYGPGQQ)₄GPGGPSGPGSAAAAAAAAAAAAAAAA (SEQ ID NO:22)(GPGGYGPGQQ)₁₆GPGGPSGPGSAAAAAAAA (SEQ ID NO:23)(GGYGPGAGQQGPGSQGPGSGGQQGPGGQ)₁ GPYGPSAAAAAAAA (SEQ ID NO:24),(GGYGPGAGQQGPGSQGPGSGGQQGPGGQ)₂ GPYGPSAAAAAAAA (SEQ ID NO:25), and(GGYGPGAGQQGPGSQGPGSGGQQGPGGQ)₃ GPYGPSAAAAAAAA (SEQ ID NO:26), andrecombinant silk protein sequences made up of the fusion proteins:MGHHHHHHHHHHSSGHIDDDDKHMLEDPP-[(GGAGPGGAGPGGAGPGGAGP)₁(GGPSGPGSAAAAAAAAGP)]₂₀—(SEQ ID NO. 27),MGHHHHHHHHHHSSGHIDDDDKHMLEDPP-[(GGAGPGGAGPGGAGPGGAGP)₂(GGPSGPGSAAAAAAAAGP)]₁₄—(SEQ ID NO:28),MGHHHHHHHHHHSSGHIDDDDKHMLEDPP-[(GGAGPGGAGPGGAGPGGAGP)₄(GGPSGPGSAAAAAAAAGP)]₈—(SEQ IDNO:29)—MGHHHHHHHHHHSSGHIDDDDKHMLEDPP-(GGAGPGGAGPGGAGPGGAGP)₄₀—(SEQ IDNO:30), MGHHHHHHHHHHSSGHIDDDDKHMLEDPP-[(GGYGPGGSGPGGYGPGGSGP)₁(GGPSGPGSAAAAAAAAGP)]₂₀—(SEQ ID NO:31),MGHHHHHHHHHHSSGHIDDDDKHMLEDPP-[(GGYGPGGSGPGGYGPGGSGP)₂(GGPSGPGSAAAAAAAAGP)]₁₄—(SEQ ID NO:32),MGHHHHHHHHHHSSGHIDDDDKHMLEDPP-[(GGYGPGGSGPGGYGPGGSGP)₄(GGPSGPGSAAAAAAAAGP)]₈—(SEQ ID NO:33) andMGHHHHHHHHHHSSGHIDDDDKHMLEDPP-(GGYGPGGSGPGGYGPGGSGP)₄₇—(SEQ ID NO:34);RPHMSRPAPAPRPLPEPLPAPRPIPAPLPRPVPIRPLPAPRGSKL (SEQ ID NO:36) which isduplicated to make proteins up to 350 kDa,RPHMTSVSQSQQASVSQSQQASVSQSQQASVSQSQQASVSQSQQSSNAYSQQAS GSKL (SEQ IDNO:38) which is duplicated to make proteins up to 350 kDa, andRPHMSRPAPAPRPLPEPLPAPRPIPAPLPRPVPIVSQVQQASIQQAQSSSAQSRQSAVAQQASVSQSQQASVSQSQQASVSQSQQASVSQSQQASVSQSQQSSNAYSAASNAASSVSQASSASSYFNSQVVQSTLSSSLQSSSALSSIAYGQTSANINDVAAAVARSVSQSLGVSQQAAQSVISQQLASAGAGASAQTLAQLISSAVSSLVQQSGTVSAGQEQSISQALSSSILSSLSQVVAQRPLPAPRGSKL (SEQ ID NO:40) which isduplicated to make proteins up to 350 kDa.

Nucleic Acid Molecules

Nucleic acid molecules encoding the polypeptides of the invention may beprepared by two general methods: (1) synthesis from appropriatenucleotide triphosphates, or (2) isolation from biological sources. Bothmethods utilize protocols well known in the art. The availability ofnucleotide sequence information, such as the DNA sequences encoding anatural or synthetic spider silk protein, enables preparation of anisolated nucleic acid molecule of one or more embodiments of the presentinvention by oligonucleotide synthesis. Synthetic oligonucleotides maybe prepared by the phosphoramidite method employed in the AppliedBiosystems 38A DNA Synthesizer or similar devices. The resultantconstruct may be used directly or purified according to methods known inthe art, such as high performance liquid chromatography (HPLC).

In accordance with at least one aspect of the present invention, nucleicacids having the appropriate level of sequence homology with sequencesencoding a spider silk protein may be identified by using hybridizationand washing conditions of appropriate stringency. Such methods areuseful for a variety of purposes, including the screening of librariescomprising mutated spider silk-encoding nucleic acid sequences fordesired properties. For example, hybridizations may be performed, usinga hybridization solution comprising: 5×SSC, 5×Denhardt's reagent, 1.0%SDS, 100 ug/ml denatured, fragmented salmon sperm DNA, 0.05% sodiumpyrophosphate and up to 50% formamide. Hybridization is carried out at37-42° C. for at least six hours. Following hybridization, filters arewashed as follows: (1) 5 minutes at room temperature in 2×SSC and 1%SDS; (2) 15 minutes at room temperature in 2×SSC and 0.1% SDS; (3) 30minutes-1 hour at 37° C. in 1×SSC and 1% SDS; (4) 2 hours at 42-65° C.in 1×SSC and 1% SDS, changing the solution every 30 minutes.

The nucleic acid molecules described herein include cDNA, genomic DNA,RNA, and fragments thereof which may be single- or double-stranded.Thus, oligonucleotides are provided having sequences capable ofhybridizing with at least one sequence of a nucleic acid sequence, suchas selected segments of sequences encoding a spider silk protein. Alsocontemplated in the scope of the present invention are methods of usefor oligonucleotide probes which specifically hybridize with DNA fromsequences encoding a spider silk protein under high stringencyconditions. Primers capable of specifically amplifying sequencesencoding a spider silk protein are also provided. As mentionedpreviously, such oligonucleotides are useful as primers for detecting,isolating and amplifying sequences encoding a spider silk protein.

Alternatively, standard purification strategies designed todifferentially isolate silk protein from plant homogenates may be usedto advantage. Purification of a plant-expressed spider silk protein maybe facilitated by its extreme stability under conditions that denaturetypical proteins, such as, for example, high heat and low pH.Accordingly, general protein purification strategies may be adapted tooptimize silk purification from leaves. Above-ground portions oftransgenic plants may be harvested and allowed to air dry as per normalproduction practices. The plant material may be homogenized in anappropriate buffer followed by various treatments designed todifferentially eliminate contaminants. Silk protein recovery may beoptimized following treatments in which plant extracts are subject toany one or a combination of the following: 1) boiling in the presence orabsence of detergent; 2) differential centrifugation; 3) progressivelydecreasing the pH; and 4) precipitation with varying concentrations ofurea or ammonium sulfate. One of ordinary skill in the art may vary theabove treatments to optimize the yield and efficiency of purification ofspider silk proteins from plants.

The quantity of silk protein may be determined by immunoblotting and thepurity and concentration assessed definitively by amino acid analysis.Purified silk protein may be analyzed for mechanical properties aspreviously described to ensure that the recombinant protein possessesthe desired properties.

A protein produced according to the present invention may be chemicallymodified after synthesis of the polypeptide. The presence of severalcarboxylic acid side chains (Asp or Glu) in the spacer regionsfacilitates the attachment of a variety of different chemical groups tosilk proteins including amino acids having such side chains. Thesimplest and easiest procedure is to use a water-soluble carbodiimide toattach the modifying group via a primary amine. If the group to beattached has no primary amine, a variety of linking agents can beattached via their own primary amines and the modifying group attachedvia an available chemistry.

Where appropriate, the DNA of interest may be optimized for increasedexpression in the transformed plant. That is, the coding sequences canbe synthesized using plant-preferred codons for improved expression.Methods are available and known to those skilled in the art forsynthesizing plant-preferred genes.

Exemplary Methods for Generation of Spider Silk Proteins

In view of the unique properties of spider silk proteins, specialconsiderations should be applied to the generation of synthetic spidersilk proteins. The repetitive nature of amino acid sequences encodingthese proteins may render synthesis of a full length spider silkprotein, or fragments thereof, technically challenging. To facilitateproduction of full length silk protein molecules, the following protocolis provided.

The polypeptides of the present invention can be made by directsynthesis or by expression from cloned DNA. Means for expressing clonedDNA are set forth above and are generally known in the art. Thefollowing considerations are recommended for the design of expressionvectors used to express DNA encoding spider silk proteins.

First, since spider silk proteins are highly repetitive in structure,cloned DNA should be propagated and expressed in host cell strains thatcan maintain repetitive sequences in extrachromosomal elements (e.g.,SURE™ cells, Stratagene). The prevalence of specific amino acids (e.g.,alanine, glycine, proline, and glutamine) also suggests that it might beadvantageous to use a host cell that over-expresses tRNA for these aminoacids or in which these specific tRNAs are known to be in highabundance.

Method for Use of Fluorescent Protein (FP) in Corn

The discovery and use of fluorescent proteins, as used herein are alsoknown as sortable markers, such as the green fluorescent protein (GFP)(SEQ ID NO:17), has revolutionized the way protein localization isperformed. Fluorescent protein (FP) fusions allow analysis of dynamiclocalization patterns in real time. Over the last several years, anumber of different colored fluorescent proteins have been developed andmay be used in various constructs of the present invention, includingyellow FP (YFP) (SEQ ID NO:19), cyan FP (CFP) (SEQ ID NO:15), red FP(mRFP) (SEQ ID NO:13), the maize specific cerulean FP (mCeFP) (SEQ IDNO:53), the mCherry maize specific FP (mChRFP) (SEQ ID NO:51), the maizespecific blue FP, TagBFP, (mBFP)(SEQ ID NO:49), the maize specific tealFP (mTFP) (SEQ ID NO: 47) and others. Some of these proteins haveimproved spectral properties, allowing analysis of fusion proteins for alonger period of time and permitting their use in photobleachingexperiments. Others are less sensitive to pH, and other physiologicalparameters, making them more suitable for use in a variety of cellularcontexts. Additionally, FP-tagged proteins can be used inprotein-protein interaction studies by bioluminescence resonance energytransfer (BRET) or fluorescence resonance energy transfer (FRET).High-throughput analyses of FP fusion proteins in Arabidopsis have beenperformed by overexpressing cDNA-GFP fusions driven by strongconstitutive promoters. Although useful, this approach has inherentlimitations, as it does not report tissue-specificity, andoverexpression of multimeric proteins may disrupt the complex.Furthermore, overexpression can lead to protein aggregation and/ormislocalization.

In order to tag a specific gene with a fluorescent protein such as thered fluorescent protein (mRFP), usually a gene ideal for tagging hasbeen identified through forward genetic analysis or by homology to aninteresting gene from another model system. For generation of nativeexpression constructs, full-length genomic sequence is required. Fortagging of the full-length gene with an FP, the full-length genesequence should be available, including all intron and exon sequences. Astandard protocol is to insert the mRFP tag or marker at a defaultposition of ten amino acids upstream of the stop codon, followingmethods known in the art established for Arabidopsis. The rationale isto avoid masking C-terminal targeting signals (such as endoplasmicreticulum (ER) retention or peroxisomal signals). In addition, byavoiding the N-terminus, disruption of N-terminal targeting sequences ortransit peptides is avoided. However, choice of tag insertion iscase-dependent, and it should be based on information on functionaldomains from database searches. If a homolog of the gene of interest hasbeen successfully tagged in another organism, this information is alsoused to choose the optimal tag insertion site. A set of four primers isdesigned for amplification of the target locus. Primers P1 and P2amplify the 5′ regulatory regions and partial coding region, extendingto the position where the mRFP tag will be inserted. The P3 and P4primers are used to amplify the remainder of the gene from the taginsertion site and including the 3′ regulatory regions. Maize genomicDNA is used for amplification of P1 to P2 and P3 to P4 fragments.However, in cases where amplification from genomic DNA fails, maize BACDNA clones, if available can be used as the PCR template. Primer designsoftware PRIMER3 is used for design of the P1-P4 primers. In general,the primer T m should be 60-62° C., but is dependent on primerrequirements. The P1 and P4 primers have linkers overlapping with theGateway™ ttPCR primers in addition to the gene-specific sequences toallow cloning of the PCR product in Gateway™ compatible vectors.Similarly the primers P2 and P3 contain gene specific sequences as wellas linkers that are complementary to sequences from the mRFP clones toallow incorporation of the mRFP tag into the ttPCR product.

A cleavable linker peptide may be placed between proteins such that theycan be cleaved and the desired protein obtained.

Transcription Terminator

The transcription termination region of the constructs of the presentinvention is a downstream regulatory region including the stop codon(TGA, TAG or TAA) and the transcription terminator sequence (SEQ IDNO:5). Alternative transcription termination regions which maybe usedmay be native with the transcriptional initiation region, may be nativewith the DNA sequence of interest, or may be derived from anothersource. The transcription termination region may be naturally occurring,or wholly or partially synthetic. Convenient transcription terminationregions are available from the Ti-plasmid of A. tumefaciens, such as theoctopine synthase and nopaline synthase transcription terminationregions or from the genes for beta-phaseolin, the chemically inducibleplant gene, pIN.

Percent Similarity and Percent Identity

Percent identity refers to the comparison of the homozygous alleles oftwo plant varieties. Percent identity is determined by comparing astatistically significant number of the homozygous alleles of twodeveloped varieties. For example, a percent identity of 90% betweenplant variety 1 and plant variety 2 means that the two varieties havethe same allele at 90% of their loci. Percent identity as used hereinwith respect to two nucleic acids refers to the comparison of the entiresequence for each of the two nucleic acids and is determined by GAPalignment using default parameters (GCG, GAP version 10, Accelrys, SanDiego, Calif.). GAP uses the algorithm of Needleman and Wunsch to findthe alignment of two complete sequences that maximizes the number ofmatches and minimizes the number of sequence gaps. Sequences which have100% identity are identical. The present invention encompasses nucleicacids that have about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% sequence identity to the specified nucleicacid.

Percent similarity refers to the comparison of the homozygous alleles ofone plant variety with those of another plant, and if the homozygousallele of the first plant matches at least one of the alleles from theother plant then they are scored as similar. Percent similarity isdetermined by comparing a statistically significant number of loci andrecording the number of loci with similar alleles as a percentage. Apercent similarity of 90% between the first plant and a second plantmeans that the first matches at least one of the alleles of the secondplant at 90% of the loci.

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of chemistry, molecular biology,microbiology, recombinant DNA, genetics, immunology, cell biology, cellculture and transgenic biology, which are within the skill of the art.

EXAMPLES OF APPLICATIONS FOR THE EXPRESSION OF SPIDER SILK

The following examples are provided to illustrate further the variousapplications of the present invention and are not intended to limit theinvention beyond the limitations set forth in the appended claims.

Example 1—Synthetic Spider Silk Protein Coding Sequence E₄S₄ of aNephila clavipes MaSp 2 Construct in Corn

In at least one embodiment of the present invention a corn shoottissue-specific regulation region encoding a DNA construct is provided,represented as CeUrr-SS, comprising a tissue promoter (SEQ ID NO:3)operably linked to a synthetic spider silk protein coding sequence (SEQID NO:20). PCR is conducted using four primers (SEQ ID NO:8; SEQ IDNO:9; SEQ ID NO:10; and SEQ ID NO:11). The gene primers permitamplification of the entire regulatory region, and gene sequence. TheDNeasy® Plant Mini genomic DNA isolation kit (QIAGEN) is used for maizegenomic DNA isolation, following manufacturer's instructions. Any methodthat produces high molecular weight genomic DNA is appropriate. GenomicDNA is eluted with TE buffer and used directly for subsequent PCRreactions. KOD Hot Start DNA polymerase (Novagen), a “proofreading”enzyme, is used for amplification of the maize genomic fragments. Theproduct from the PCR is then cloned using the Gateway® system into adonor vector (available from Invitrogen).

Regardless of the cloning procedure, the final construct is thentransferred by electroporation into binary destination vectors such asan Agrobacterium plasmid or Ti plasmid and ultimately transformed intomaize. Binary plasmids are transferred to Agrobacterium (e.g., EHA101strain) by electroporation. After electroporation, 800 μL of LB mediumare added to the tubes and incubated at 28° C. for 2h with shaking.Aliquots of 50 μL and 200 μL are plated on LB plates containingspectinomycin (100 mg/L), kanamycin (50 mg/L), and chloramphenicol (25mg/L) and incubated for 2-3 days at 28° C. Spectinomycin is used forselecting the binary plasmid, whereas the other two antibiotics are forthe selection of the EHA101 Agrobacterium strain. Single colonies arepicked and grown for 2 to 3 days in 6 mL LB medium supplemented withabove antibiotics with shaking at 28° C. To verify the clones, theplasmids are isolated from these cultures by a modified alkaline lysismethod and checked by restriction enzyme digestion or PCR. Followingclone verification, the constructs are transformed into maize togenerate stable lines. Transgenic maize plants expressing the syntheticspider silk protein genes are generated. Maize transformants areprovided as seedlings on sterile Petri plates, regenerated from callustissue from Hill lines (classified here as T₀ generation). The plantsare transferred from plates to growth chambers maintained at 25-28° C.(16-h light period) until the roots and shoots are several centimeterslong. Once acclimated in the growth chamber, the first generationseedlings are screened for expression. The seedlings are transferred tosoil in small pots and covered with a plastic dome to maintain humidityfor 3-4 days and encourage optimal root growth. The establishedseedlings are then transferred to larger pots for growth and pollinationin the greenhouse. To maintain adequate growth, greenhouse conditionsare optimized for maize.

Example 2—Synthetic Spider Silk Protein Coding Sequence E₄S₄ of aNephila clavipes MaSp 2 Construct in Corn

In at least one embodiment of the present invention a corn endospermtissue regulation region encoding a DNA construct is provided,represented as CeUrr-SS, comprising a tissue promoter encoded with aleader sequence (SEQ ID NO:1) operably linked to a synthetic spider silkprotein coding sequence (SEQ ID NO:20). PCR is conducted using fourprimers (SEQ ID NO:8; SEQ ID NO:9; SEQ ID NO:10; and SEQ ID NO:11). Thegene specific primers permit amplification of the entire regulatoryregion, gene sequence. The DNeasy® Plant Mini genomic DNA isolation kit(QIAGEN) is used for maize genomic DNA isolation, followingmanufacturer's instructions. Any method that produces high molecularweight genomic DNA is appropriate. Genomic DNA is eluted with TE bufferand used directly for subsequent PCR reactions. KOD Hot Start DNApolymerase (Novagen), a “proofreading” enzyme, is used for amplificationof the maize genomic fragments. The product from the PCR is then clonedusing the Gateway® system into a donor vector (available fromInvitrogen).

Regardless of the cloning procedure, the final construct is thentransferred by electroporation into binary destination vectors such asan Agrobacterium plasmid or Ti plasmid and ultimately transformed intomaize. Binary plasmids are transferred to Agrobacterium (e.g., EHA101strain) by electroporation. Binary plasmids are transferred toAgrobacterium (e.g., EHA101 strain) by electroporation. Afterelectroporation, 800 μL of LB medium are added to the tubes andincubated at 28° C. for 2 h with shaking. Aliquots of 50 μL and 200 μLare plated on LB plates containing spectinomycin (100 mg/L), kanamycin(50 mg/L), and chloramphenicol (25 mg/L) and incubated for 2-3 days at28° C. Spectinomycin is used for selecting the binary plasmid, whereasthe other two antibiotics are for the selection of the EHA101Agrobacterium strain. Single colonies are picked and grown for 2 to 3days in 6 mL LB medium supplemented with above antibiotics with shakingat 28° C. To verify the clones, the plasmids are isolated from thesecultures by a modified alkaline lysis method and checked by restrictionenzyme digestion or PCR. Following clone verification, the constructsare transformed into maize to generate stable lines. Transgenic maizeplants expressing the synthetic spider silk protein genes are generated.Maize transformants are provided as seedlings on sterile Petri plates,regenerated from callus tissue from Hill lines (classified here as T₀generation). The plants are transferred from plates to growth chambersmaintained at 25-28° C. (16-h light period) until the roots and shootsare several centimeters long. Once acclimated in the growth chamber, thefirst generation seedlings are screened for expression. The seedlingsare transferred to soil in small pots and covered with a plastic dome tomaintain humidity for 3-4 days and encourage optimal root growth. Theestablished seedlings are then transferred to larger pots for growth andpollination in the greenhouse. To maintain adequate growth, greenhouseconditions are optimized for maize.

Example 3—Synthetic Spider Silk Protein Coding Sequence E₄S₄ of aNephila clavipes MaSp 2 Construct in Corn with a Terminator Sequence

In at least one embodiment of the present invention a corn endospermtissue regulation region encoding a DNA construct is provided,represented as CeUrr-SS-X, comprising a promoter (SEQ ID NO:3) operablylinked to a synthetic spider silk protein coding sequence (SEQ ID NO:20)which is operably linked to a transcription terminator sequence (SEQ IDNO:5). A PCR is conducted using four primers (SEQ ID NO:8; SEQ ID NO:9;SEQ ID NO:10; and SEQ ID NO:11). The gene specific primers permitamplification of the entire regulatory region, gene sequence. TheDNeasy® Plant Mini genomic DNA isolation kit (QIAGEN) is used for maizegenomic DNA isolation, following manufacturer's instructions. Any methodthat produces high molecular weight genomic DNA is appropriate. GenomicDNA is eluted with TE buffer and used directly for subsequent PCRreactions. KOD Hot Start DNA polymerase (Novagen), a “proofreading”enzyme, is used for amplification of the maize genomic fragments. Theproduct from the PCR is then cloned using the Gateway® system into adonor vector (available from Invitrogen).

Regardless of the cloning procedure, the final construct is thentransferred by electroporation into binary destination vectors such asan Agrobacterium plasmid or Ti plasmid and ultimately transformed intomaize. Binary plasmids are transferred to Agrobacterium (e.g., EHA101strain) by electroporation. After electroporation, 800 μL of LB mediumare added to the tubes and incubated at 28° C. for 2 h with shaking.Aliquots of 50 μL and 200 μL are plated on LB plates containingspectinomycin (100 mg/L), kanamycin (50 mg/L), and chloramphenicol (25mg/L) and incubated for 2-3 days at 28° C. Spectinomycin is used forselecting the binary plasmid, whereas the other two antibiotics are forthe selection of the EHA101 Agrobacterium strain. Single colonies arepicked and grown for 2 to 3 days in 6 mL LB medium supplemented withabove antibiotics with shaking at 28° C. To verify the clones, theplasmids are isolated from these cultures by a modified alkaline lysismethod and checked by restriction enzyme digestion or PCR. Followingclone verification, the constructs are transformed into maize togenerate stable lines. Transgenic maize plants expressing the syntheticspider silk protein genes are generated. Maize transformants areprovided as seedlings on sterile Petri plates, regenerated from callustissue from Hill lines (classified here as T₀ generation). The plantsare transferred from plates to growth chambers maintained at 25-28° C.(16-h light period) until the roots and shoots are several centimeterslong. Once acclimated in the growth chamber, the first generationseedlings are screened for expression. The seedlings are transferred tosoil in small pots and covered with a plastic dome to maintain humidityfor 3-4 days and encourage optimal root growth. The establishedseedlings are then transferred to larger pots for growth and pollinationin the greenhouse. To maintain adequate growth, greenhouse conditionsare optimized for maize.

Example 4—Synthetic Spider Silk Protein Coding Sequence E₄S₄ of aNephila clavipes MaSp 2 Construct in Corn Using the Multisite Gateway®Cloning Procedure

In at least one embodiment of the present invention a corn endospermtissue regulation region encoding a DNA construct is provided,represented as CeUrr-SS-X, comprising a promoter (SEQ ID NO:3) operablylinked to a synthetic spider silk protein coding sequence (SEQ ID NO:20)which is operably linked to a transcription terminator sequence (SEQ IDNO:5). Using the Multisite Gateway® cloning procedure, available fromPCR products with Gateway® entry sites and corresponding to the 5′UTRplus gene ORF (for C-terminal tagging) and 3′UTR plus gene ORF (forN-terminal tagging), are cloned into pDONR vectors using the Gateway® BPreaction system. An expression clone is generated by combining entryclones, including a fluorescent tag entry vector, along with thepTF101.1 maize binary vector that has been converted into a 3-wayGateway® destination vector.

Regardless of the cloning procedure, the final construct is thentransferred by electroporation into binary destination vectors such asan Agrobacterium plasmid or Ti plasmid and ultimately transformed intomaize. Binary plasmids are transferred to Agrobacterium (e.g., EHA101strain) by electroporation. After electroporation, 800 μL of LB mediumare added to the tubes and incubated at 28° C. for 2 h with shakingAliquots of 50 μL and 200 μL are plated on LB plates containingspectinomycin (100 mg/L), kanamycin (50 mg/L), and chloramphenicol (25mg/L) and incubated for 2-3 days at 28° C. Spectinomycin is used forselecting the binary plasmid, whereas the other two antibiotics are forthe selection of the EHA101 Agrobacterium strain. Single colonies arepicked and grown for 2 to 3 days in 6 mL LB medium supplemented withabove antibiotics with shaking at 28° C. To verify the clones, theplasmids are isolated from these cultures by a modified alkaline lysismethod and checked by restriction enzyme digestion or PCR. Followingclone verification, the constructs are transformed into maize togenerate stable lines. Transgenic maize plants expressing the syntheticspider silk protein genes are generated. Maize transformants areprovided as seedlings on sterile Petri plates, regenerated from callustissue from Hill lines (classified here as T₀ generation). The plantsare transferred from plates to growth chambers maintained at 25-28° C.(16-h light period) until the roots and shoots are several centimeterslong. Once acclimated in the growth chamber, the first generationseedlings are screened for expression. The seedlings are transferred tosoil in small pots and covered with a plastic dome to maintain humidityfor 3-4 days and encourage optimal root growth. The establishedseedlings are then transferred to larger pots for growth and pollinationin the greenhouse. To maintain adequate growth, greenhouse conditionsare optimized for maize.

Example 5—Identification of Synthetic Spider Silk Protein CodingSequence Using a Red Fluorescent Protein

In at least one embodiment of the present invention provides a cornendosperm tissue regulation region encoding a DNA construct, representedas CeUrr-SS-FP-X, comprising a promoter (SEQ ID NO:3) operably linked toa synthetic spider silk protein coding sequence (SEQ ID NO:20), operablylinked to a fluorescent protein coding sequence (SEQ ID NO:13) which isoperably linked to a transcription terminator sequence (SEQ ID NO:5). APCR is conducted using four primers (SEQ ID NO:8; SEQ ID NO:9; SEQ IDNO:10; and SEQ ID NO:11). The gene specific primers permit amplificationof the entire regulatory region, gene sequence. The DNeasy® Plant Minigenomic DNA isolation kit (QIAGEN) is used for maize genomic DNAisolation, following manufacturer's instructions. Any method thatproduces high molecular weight genomic DNA is appropriate. Genomic DNAis eluted with TE buffer and used directly for subsequent PCR reactions.KOD Hot Start DNA polymerase (Novagen), a “proofreading” enzyme, is usedfor amplification of the maize genomic fragments. The product from thePCR is then cloned using the Gateway® system into a donor vector(available from Invitrogen).

For gene tagging, mRFP1 (SEQ ID NO:13) fluorescent protein tags aremodified to remove start and stop codons and add flexible linkerpeptides flanking the ends, allowing them to be used as either C- orN-terminal fusions, or as internal fusions. These flexible linkers helpto minimize folding interference between the target protein and thefluorescent protein. In addition, the linker peptide sequences containan FseI site at the 5′ end and a SfiI site at the 3′ end. Theserestriction enzyme sites can be used to replace one fluorescent proteintag with another, or for addition of others, such as affinitypurification tags for proteomics. mRFP1 clones are generated with theselinkers. The fluorescent protein tag fragments are PCR amplified fromthe above plasmids using the following primers of SEQ ID NO:41 and SEQID NO:42.

Regardless of the cloning procedure, the final construct is thentransferred by electroporation into binary destination vectors such asan Agrobacterium plasmid or Ti plasmid and ultimately transformed intomaize. Binary plasmids are transferred to Agrobacterium (e.g., EHA101strain) by electroporation. After electroporation, 800 μL of LB mediumare added to the tubes and incubated at 28° C. for 2 h with shaking.Aliquots of 50 μL and 200 μL are plated on LB plates containingspectinomycin (100 mg/L), kanamycin (50 mg/L), and chloramphenicol (25mg/L) and incubated for 2-3 days at 28° C. Spectinomycin is used forselecting the binary plasmid, whereas the other two antibiotics are forthe selection of the EHA101 Agrobacterium strain. Single colonies arepicked and grown for 2 to 3 days in 6 mL LB medium supplemented withabove antibiotics with shaking at 28° C. To verify the clones, theplasmids are isolated from these cultures by a modified alkaline lysismethod and checked by restriction enzyme digestion or PCR. Followingclone verification, the constructs are transformed into maize togenerate stable lines. Transgenic maize plants expressing the FP taggedgenes are generated. Maize transformants are provided as seedlings onsterile Petri plates, regenerated from callus tissue from Hill lines(classified here as T₀ generation). The plants are transferred fromplates to growth chambers maintained at 25-28° C. (16-h light period)until the roots and shoots are several centimeters long. Once acclimatedin the growth chamber, the first generation seedlings are screened forexpression. The seedlings are transferred to soil in small pots andcovered with a plastic dome to maintain humidity for 3-4 days andencourage optimal root growth. The established seedlings are thentransferred to larger pots for growth and pollination in the greenhouse.To maintain adequate growth, greenhouse conditions are optimized formaize.

The mRFP will produce a red fluorescence in the presence of UV light,thereby allowing for the monitoring of the synthetic spider silk proteinactivity and the presence or absence of the tagged protein in a targetedregion. The expression of the spider silk is localized to corn kernelendosperm and will emit a red fluorescence in the presence of UV light.Sunlight is sufficient to excite the fluorescence so that the kernelscontaining the transgene appear pink. The mRFP will eventually bedenatured during use or treatment of the silk and so the silk wouldlikely lose the pink color.

Example 6—Synthetic Spider Silk Protein Coding Sequence E₄S₈ in CornIdentified Using a Cyan Fluorescent Protein

Example five is repeated with the exception for gene tagging, CFP (SEQID NO:15) fluorescent protein tags are modified to remove start and stopcodons and add flexible linker peptides flanking the ends, allowing themto be used as either C- or N-terminal fusions, or as internal fusions.These flexible linkers help to minimize folding interference between thetarget protein and the fluorescent protein. In addition, the linkerpeptide sequences contain an FseI site at the 5′ end and a SfiI site atthe 3′ end. These restriction enzyme sites can be used to replace onefluorescent protein tag with another, or for addition of others, such asaffinity purification tags for proteomics. CFP clones are generated withthese linkers. The fluorescent protein tag fragments are PCR amplifiedfrom the above plasmids using the following primers of SEQ ID NO:43 andSEQ ID NO:44.

Example 7—Synthetic Spider Silk Protein Coding Sequence E₄S₁₆ in CornIdentified with a Yellow Fluorescent Protein

Example five is repeated with the exception of gene tagging. YFP (SEQ IDNO:19) fluorescent protein tags are modified to remove start and stopcodons and add flexible linker peptides flanking the ends, allowing themto be used as either C- or N-terminal fusions, or as internal fusions.These flexible linkers help to minimize folding interference between thetarget protein and the fluorescent protein. In addition, the linkerpeptide sequences contain an FseI site at the 5′ end and a SfiI site atthe 3′ end. These restriction enzyme sites can be used to replace onefluorescent protein tag with another, or for addition of others, such asaffinity purification tags for proteomics. YFP clones are generated withthese linkers. The fluorescent protein tag fragments are PCR amplifiedfrom the above plasmids using the following primers of SEQ ID NO:43 andSEQ ID NO:44.

Example 8—Identification of Synthetic Spider Silk Protein CodingSequence Using a Maize Specific Fluorescent Protein

Example five is repeated with the exception for gene tagging. CeruleanFP (SEQ ID NO:52) fluorescent protein tags are modified to remove startand stop codons and add flexible linker peptides flanking the ends,allowing them to be used as either C- or N-terminal fusions, or asinternal fusions. These flexible linkers help to minimize foldinginterference between the target protein and the fluorescent protein. Inaddition, the linker peptide sequences contain an FseI site at the 5′end and a SfiI site at the 3′ end. These restriction enzyme sites can beused to replace one fluorescent protein tag with another, or foraddition of others, such as affinity purification tags for proteomics.Cerulean clones are generated with these linkers. The fluorescentprotein tag fragments are PCR amplified from the above plasmids usingthe following primers of SEQ ID NO:59 and SEQ ID NO:60.

Example 9—Identification of Synthetic Spider Silk Protein CodingSequence Using a Maize Specific Cherry Fluorescent Protein

Example five is repeated with the exception for gene tagging. Maizespecific cherry (SEQ ID NO:51) fluorescent protein tags are modified toremove start and stop codons and add flexible linker peptides flankingthe ends, allowing them to be used as either C- or N-terminal fusions,or as internal fusions. These flexible linkers help to minimize foldinginterference between the target protein and the fluorescent protein. Inaddition, the linker peptide sequences contain an FseI site at the 5′end and a SfiI site at the 3′ end. These restriction enzyme sites can beused to replace one fluorescent protein tag with another, or foraddition of others, such as affinity purification tags for proteomics.mCHERRY clones are generated with these linkers. The fluorescent proteintag fragments are PCR amplified from the above sequences using thefollowing primers of SEQ ID NO:57 and SEQ ID NO:58.

Example 10—Identification of Synthetic Spider Silk Protein CodingSequence Using a Maize Specific Blue Fluorescent Protein

Example five is repeated with the exception of for gene tagging. Maizespecific blue fluorescent protein (SEQ ID NO:48) or (SEQ ID NO:49) tagsare modified to remove start and stop codons and add flexible linkerpeptides flanking the ends, allowing them to be used as either C- orN-terminal fusions, or as internal fusions. These flexible linkers helpto minimize folding interference between the target protein and thefluorescent protein. In addition, the linker peptide sequences containan FseI site at the 5′ end and a SfiI site at the 3′ end. Theserestriction enzyme sites can be used to replace one fluorescent proteintag with another, or for addition of others, such as affinitypurification tags for proteomics. The maize specific blue FP clones aregenerated with these linkers. The fluorescent protein tag fragments arePCR amplified from the above sequences using the following primers ofSEQ ID NO:57 and SEQ ID NO:58.

Example 11—Identification of Synthetic Spider Silk Protein CodingSequence Using a Maize Specific Teal Fluorescent Protein

Example five is repeated with the exception for gene tagging. The maizeteal FP (SEQ ID NO:47) fluorescent protein tags are modified to removestart and stop codons and add flexible linker peptides flanking theends, allowing them to be used as either C- or N-terminal fusions, or asinternal fusions. These flexible linkers help to minimize foldinginterference between the target protein and the fluorescent protein. Inaddition, the linker peptide sequences contain an FseI site at the 5′end and a SfiI site at the 3′ end. These restriction enzyme sites can beused to replace one fluorescent protein tag with another, or foraddition of others, such as affinity purification tags for proteomics.Teal clones are generated with these linkers. The fluorescent proteintag fragments are PCR amplified from the above plasmids using thefollowing primers of SEQ ID NO:57 and SEQ ID NO:58

Example 12—Identification of Synthetic Spider Silk Protein CodingSequence in Corn Shoot Tissue Using a Red Fluorescent Protein

In at least one embodiment of the present invention provides a cornshoot tissue regulation region encoding a DNA construct, represented asPsUrr-SS-FP-X comprising a corn shoot tissue promoter (SEQ ID NO:62)operably linked to a synthetic spider silk protein coding sequence (SEQID NO:20), operably linked to a fluorescent protein coding sequence (SEQID NO:13) and operably linked to a transcription terminator sequence. APCR is conducted using two primers (SEQ ID NO:63 and SEQ ID NO:64). Thegene specific primers permit amplification of the entire regulatoryregion, gene sequence. The DNeasy® Plant Mini genomic DNA isolation kit(QIAGEN) is used for maize genomic DNA isolation, followingmanufacturer's instructions. Any method that produces high molecularweight genomic DNA is appropriate. Genomic DNA is eluted with TE bufferand used directly for subsequent PCR reactions. KOD Hot Start DNApolymerase (Novagen), a “proofreading” enzyme, is used for amplificationof the maize genomic fragments. The product from the PCR is then clonedusing the Gateway® system into a donor vector.

For gene tagging, mRFP1 (SEQ ID NO:13) fluorescent protein tags aremodified to remove start and stop codons and add flexible linkerpeptides flanking the ends, allowing them to be used as either C- orN-terminal fusions, or as internal fusions. These flexible linkers helpto minimize folding interference between the target protein and thefluorescent protein. In addition, the linker peptide sequences containan FseI site at the 5′ end and a SfiI site at the 3′ end. Theserestriction enzyme sites can be used to replace one fluorescent proteintag with another, or for addition of others, such as affinitypurification tags for proteomics. mRFP1 clones are generated with theselinkers. The fluorescent protein tag fragments are PCR amplified fromthe above plasmids using the following primers of SEQ ID NO:41 and SEQID NO:42.

Regardless of the cloning procedure, the final construct is thentransferred by electroporation into binary destination vectors such asan Agrobacterium plasmid or Ti plasmid and ultimately transformed intomaize. Binary plasmids are transferred to Agrobacterium (e.g., EHA101strain) by electroporation. After electroporation, 800 μL of LB mediumare added to the tubes and incubated at 28° C. for 2 h with shaking.Aliquots of 50 μL and 200 μL are plated on LB plates containingspectinomycin (100 mg/L), kanamycin (50 mg/L), and chloramphenicol (25mg/L) and incubated for 2-3 days at 28° C. Spectinomycin is used forselecting the binary plasmid, whereas the other two antibiotics are forthe selection of the EHA101 Agrobacterium strain. Single colonies arepicked and grown for 2 to 3 days in 6 mL LB medium supplemented withabove antibiotics with shaking at 28° C. To verify the clones, theplasmids are isolated from these cultures by a modified alkaline lysismethod and checked by restriction enzyme digestion or PCR. Followingclone verification, the constructs are transformed into maize togenerate stable lines. Transgenic maize plants expressing the FP taggedgenes are generated. Maize transformants are provided as seedlings onsterile Petri plates, regenerated from callus tissue from Hill lines(classified here as T₀ generation). The plants are transferred fromplates to growth chambers maintained at 25-28° C. (16-h light period)until the roots and shoots are several centimeters long. Once acclimatedin the growth chamber, the first generation seedlings are screened forexpression. The seedlings are transferred to soil in small pots andcovered with a plastic dome to maintain humidity for 3-4 days andencourage optimal root growth. The established seedlings are thentransferred to larger pots for growth and pollination in the greenhouse.To maintain adequate growth, greenhouse conditions are optimized formaize.

The mRFP will produce a red fluorescence in the presence of UV light,thereby allowing for the monitoring of the synthetic spider silk proteinactivity and the presence or absence of the tagged protein in a targetedregion. The expression of the spider silk is localized to corn shootsand will emit a red fluorescence in the presence of UV light. Sunlightis sufficient to excite the fluorescence so that the shoots containingthe transgene appear pink. The mRFP will eventually be denatured duringuse or treatment of the silk and so the silk would likely lose the pinkcolor.

Example 13—Identification of Synthetic Spider Silk Protein CodingSequence in Corn Shoot Tissue Using a Red Fluorescent Protein

Example thirteen is repeated with the exception of a corn shoot tissueregulation region encoding a DNA construct represented as PsUrr-SS-FP-Xwhich includes a corn shoot promoter (SEQ ID NO:61) operably linked to asynthetic spider silk protein coding sequence (SEQ ID NO:20) andoperably linked to a transcription terminator sequence. A PCR isconducted using two primers (SEQ ID NO:63 and SEQ ID NO:64).

Example 14—Identification of Synthetic Spider Silk Protein CodingSequence in Corn Shoot Tissue Using a Red Fluorescent Protein

Example thirteen is repeated with the exception of a corn shoot tissueregulation region encoding a DNA construct represented as PsUrr-SS-FP-Xwhich includes a corn shoot promoter (SEQ ID NO:62) operably linked to asynthetic spider silk protein coding sequence (SEQ ID NO:20) operablylinked to a fluorescent protein coding sequence (SEQ ID NO:13) andoperably linked to a transcription terminator sequence. A PCR isconducted using two primers (SEQ ID NO:63 and SEQ ID NO:64.

Example 15—Synthetic Spider Silk Protein Coding Sequence E₁₆S₈ of aNephila clavipes MaSp 2 Construct in Corn

Example two is repeated with the exception of a corn endosperm tissueregulation region encoding a DNA construct represented as CeUrr-SS-FP-Xwhich includes a promoter (SEQ ID NO:3) operably linked to a syntheticspider silk protein coding sequence, E₁₆S₈ (SEQ ID NO. 23) operablylinked to a fluorescent protein coding sequence (SEQ ID NO:13) which isoperably linked to transcription terminator sequence (SEQ ID NO:4).

Example 16—Spider Silk Protein Coding Sequence E₁S₈ of an Argiope sp.MaSp 2 Construct in Corn

Example two is repeated with the exception of a corn endosperm tissueregulation region encoding a DNA construct represented as CeUrr-SS-FP-Xwhich includes a promoter (SEQ ID NO:3) operably linked to a syntheticspider silk protein coding sequence, E₁S₈ (SEQ ID NO:24), operablylinked to a fluorescent protein coding sequence (SEQ ID NO:13) which isoperably linked to a transcription terminator sequence (SEQ ID NO:5).

Example 17—Synthetic Spider Silk Protein Coding Sequence E₂S₈ of anArgiope sp. MaSp 2 Construct in Corn

Example two is repeated with the exception of a corn endosperm tissueregulation region encoding a DNA construct represented as CeUrr-SS-FP-Xwhich includes a promoter (SEQ ID NO:3) operably linked to a syntheticspider silk protein coding sequence, E₂S₈ (SEQ ID NO:25), operablylinked to a fluorescent protein coding sequence (SEQ ID NO:13) which isoperably linked to a transcription terminator sequence (SEQ ID NO:5).

Example 18—Synthetic Spider Silk Protein Coding Sequence E₃S₈ of anArgiope sp. MaSp 2 Construct in Corn

Example two is repeated with the exception of a corn endosperm tissueregulation region encoding a DNA construct represented as CeUrr-SS-FP-Xwhich includes a promoter (SEQ ID NO:3) operably linked to a syntheticspider silk protein coding sequence, E₃S₈ (SEQ ID NO:26), operablylinked to a fluorescent protein coding sequence (SEQ ID NO:13) which isoperably linked to a transcription terminator sequence (SEQ ID NO:5).

Example 19—Synthetic Spider Silk Protein Coding Sequence A1S8₂₀ in Corn

Example two is repeated with the exception of a corn endosperm tissueregulation region encoding a DNA construct represented as CeUrr-SS-FP-Xincluding a promoter (SEQ ID NO:3) operably linked to a synthetic spidersilk protein coding sequence, A1S8₂₀ (SEQ ID NO:27), operably linked toa fluorescent protein coding sequence (SEQ ID NO:13) which is operablylinked to a transcription terminator sequence (SEQ ID NO:5).

Example 20—Synthetic Spider Silk Protein Coding Sequence A1S8₁₄ in Corn

Example two is repeated with the exception of a corn endosperm tissueregulation region encoding a DNA construct represented as CeUrr-SS-FP-Xincluding a promoter (SEQ ID NO:3) operably linked to a synthetic spidersilk protein coding sequence, A1S8₁₄ (SEQ ID NO:28), operably linked toa fluorescent protein coding sequence (SEQ ID NO:13) which is operablylinked to a transcription terminator sequence (SEQ ID NO:5).

Example 21—Synthetic Spider Silk Protein Coding Sequence A1S8₈ in Corn

Example two is repeated with the exception of a corn endosperm tissueregulation region encoding a DNA construct represented as CeUrr-SS-FP-Xincluding a promoter (SEQ ID NO:3) operably linked to a synthetic spidersilk protein coding sequence, A1S8₈ (SEQ ID NO:29), operably linked to afluorescent protein coding sequence (SEQ ID NO:13) which is operablylinked to a transcription terminator sequence (SEQ ID NO:5).

Example 22—Synthetic Spider Silk Protein Coding Sequence A₄₀ in Corn

Example two is repeated with the exception of a corn endosperm tissueregulation region encoding a DNA construct represented as CeUrr-SS-FP-Xincluding a promoter (SEQ ID NO:3) operably linked to a synthetic spidersilk protein coding sequence, A₄₀ (SEQ ID NO:30), operably linked to afluorescent protein coding sequence (SEQ ID NO:13) which is operablylinked to a transcription terminator sequence (SEQ ID NO:5).

Example 23—Synthetic Spider Silk Protein Coding Sequence Y1S8₂₀ in Corn

Example two is repeated with the exception of a corn endosperm tissueregulation region encoding a DNA construct represented as CeUrr-SS-FP-Xincluding a promoter (SEQ ID NO:3) operably linked to a synthetic spidersilk protein coding sequence, Y1S8₂₀ (SEQ ID NO:31), operably linked toa fluorescent protein coding sequence (SEQ ID NO:13) which is operablylinked to a transcription terminator sequence (SEQ ID NO:5).

Example 24—Synthetic Spider Silk Protein Coding Sequence Y1S8₁₄ in Corn

Example two is repeated with the exception of a corn endosperm tissueregulation region encoding a DNA construct represented as CeUrr-SS-FP-Xincluding a promoter (SEQ ID NO:3) operably linked to a synthetic spidersilk protein coding sequence, Y1S8₁₄ (SEQ ID NO:32), operably linked toa fluorescent protein coding sequence (SEQ ID NO:13) which is operablylinked to a transcription terminator sequence (SEQ ID NO:5).

Example 25—Synthetic Spider Silk Protein Coding Sequence Y1S8₈ in Corn

Example two is repeated with the exception of a corn endosperm tissueregulation region encoding a DNA construct represented as CeUrr-SS-FP-Xincluding a promoter (SEQ ID NO:3) operably linked to a synthetic spidersilk protein coding sequence, Y1S8₈ (SEQ ID NO:33), operably linked to afluorescent protein coding sequence (SEQ ID NO:13) which is operablylinked to a transcription terminator sequence (SEQ ID NO:5).

Example 26—Synthetic Spider Silk Protein Coding Sequence Y₄₇ in Corn

Example two is repeated with the exception of a corn endosperm tissueregulation region encoding a DNA construct represented as CeUrr-SS-FP-Xincluding a promoter (SEQ ID NO:3) operably linked to a synthetic spidersilk protein coding sequence, Y₄₇ (SEQ ID NO:34), operably linked to afluorescent protein coding sequence (SEQ ID NO:13) which is operablylinked to a transcription terminator sequence (SEQ ID NO:5).

Example 27—Synthetic Spider Silk Protein Coding Sequence PXP Corn

Example two is repeated with the exception of a corn endosperm tissueregulation region encoding a DNA construct represented as CeUrr-SS-FP-Xincluding a promoter (SEQ ID NO:3) operably linked to a synthetic spidersilk protein coding sequence, PXP (SEQ ID NO:35), operably linked to afluorescent protein coding sequence (SEQ ID NO:13) which is operablylinked to a transcription terminator sequence (SEQ ID NO:5). The PXPsequence is a repeat sequence which is duplicated to make proteins up to350 kDa.

Example 28—Synthetic Spider Silk Coding Sequence PXP in Corn

Example two is repeated with the exception of a corn endosperm tissueregulation region encoding a DNA construct represented as CeUrr-SS-FP-Xincluding a promoter (SEQ ID NO:3) operably linked to a synthetic spidersilk nucleic acid coding sequence, PXP (SEQ ID NO:36), operably linkedto a fluorescent protein coding sequence (SEQ ID NO:13) which isoperably linked to a transcription terminator sequence (SEQ ID NO:5).The PXP sequence is a repeat sequence which is duplicated to makeproteins up to 350 kDa.

Example 29—Synthetic Spider Silk Protein Coding Sequence QQ in Corn

Example two is repeated with the exception of a corn endosperm tissueregulation region encoding a DNA construct represented as CeUrr-SS-FP-Xincluding a promoter (SEQ ID NO:3) operably linked to a synthetic spidersilk protein coding sequence, QQ (SEQ ID NO:37), operably linked to afluorescent protein coding sequence (SEQ ID NO:13) which is operablylinked to a transcription terminator sequence (SEQ ID NO:5). The QQsequence is a repeat sequence which is duplicated to make proteins up to350 kDa.

Example 30—Synthetic Spider Silk Coding Sequence QQ in Corn

Example two is repeated with the exception of a corn endosperm tissueregulation region encoding a DNA construct represented as CeUrr-SS-FP-Xincluding a promoter (SEQ ID NO:3) operably linked to a synthetic spidersilk nucleic acid coding sequence, QQ (SEQ ID NO:38), operably linked toa fluorescent protein coding sequence (SEQ ID NO:13) which is operablylinked to a transcription terminator sequence (SEQ ID NO:5). The QQsequence is a repeat sequence which is duplicated to make proteins up to350 kDa.

Example 31—Synthetic Spider Silk Protein Coding the Full PiriformSequence in Corn

Example two is repeated with the exception of a corn endosperm tissueregulation region encoding a DNA construct represented as CeUrr-SS-FP-Xincluding a promoter (SEQ ID NO:3) operably linked to a synthetic spidersilk protein coding full piriform sequence (SEQ ID NO:39), operablylinked to a fluorescent protein coding sequence (SEQ ID NO:13) which isoperably linked to a transcription terminator sequence (SEQ ID NO:5).The full piriform sequence is a repeat sequence which is duplicated tomake proteins up to 350 kDa.

Example 32—Synthetic Spider Silk Coding Sequence the Full PiriformSequence in Corn

Example two is repeated with the exception of a corn endosperm tissueregulation region encoding a DNA construct represented as CeUrr-SS-FP-Xincluding a promoter (SEQ ID NO:3) operably linked to a synthetic spidersilk nucleic acid coding full piriform sequence (SEQ ID NO:40), operablylinked to a fluorescent protein coding sequence (SEQ ID NO:13) which isoperably linked to a transcription terminator sequence (SEQ ID NO:5).The full piriform sequence is a repeat sequence which is duplicated tomake proteins up to 350 kDa.

Example 33—Synthetic Spider Silk Coding Protein Sequence in Corn

Example two is repeated with the exception of a corn endosperm tissueregulation region encoding a DNA construct represented as CeUrr-SS-FP-Xincluding a promoter (SEQ ID NO:2) operably linked to a synthetic spidersilk protein sequence (SEQ ID NO:20), operably linked to a fluorescentprotein coding sequence (SEQ ID NO:13) which is operably linked to atranscription terminator sequence (SEQ ID NO:5).

Example 34—Synthetic Spider Silk Coding Protein Sequence

Example two is repeated with the exception of a corn endosperm tissueregulation region encoding a DNA construct represented as CeUrr-SS-FP-Xincluding a promoter (SEQ ID NO:4) operably linked to a synthetic spidersilk protein sequence (SEQ ID NO:20), operably linked to a fluorescentprotein coding sequence (SEQ ID NO:13) which is operably linked to atranscription terminator sequence (SEQ ID NO:5).

Example 35—Synthetic Spider Silk Coding Protein Sequence

Example two is repeated with the exception of a plant endosperm tissueregulation region encoding a DNA construct represented as PeUrr-SS-FP-Xincluding a promoter (SEQ ID NO:6) operably linked to a synthetic spidersilk protein sequence (SEQ ID NO:20), operably linked to a fluorescentprotein coding sequence (SEQ ID NO:13) which is operably linked to atranscription terminator sequence (SEQ ID NO:5).

Example 36—Synthetic Spider Silk Coding Protein Sequence in BarleyEndosperm

Example two is repeated with the exception of a barley endosperm tissueregulation region encoding a DNA construct represented as PeUrr-SS-FP-Xincluding a promoter (SEQ ID NO:7) operably linked to a synthetic spidersilk protein sequence (SEQ ID NO:20), operably linked to a fluorescentprotein coding sequence (SEQ ID NO:13) which is operably linked to atranscription terminator sequence (SEQ ID NO:5).

Example 37—Synthetic Spider Silk Coding Protein Sequence in Plant LeafTissue

Example two is repeated with the exception of a tobacco leaf tissueregulation region encoding a DNA construct represented as PsUrr-SS-FP-Xincluding a promoter (SEQ ID NO:54) operably linked to a syntheticspider silk protein sequence (SEQ ID NO:20), operably linked to afluorescent protein coding sequence (SEQ ID NO:13) which is operablylinked to a transcription terminator sequence.

Example 38—Synthetic Spider Silk Coding Protein Sequence in Plant LeafTissue

Example two is repeated with the exception of a tobacco leaf tissueregulation region encoding a DNA construct represented as PsUrr-SS-FP-Xincluding a promoter (SEQ ID NO:55) operably linked to a syntheticspider silk protein sequence (SEQ ID NO:20), operably linked to afluorescent protein (SEQ ID NO:13) which is operably linked to atranscription terminator sequence.

Example 39—Synthetic Spider Silk Coding Protein Sequence in Plant LeafTissue

Example two is repeated with the exception of a tobacco leaf tissueregulation region encoding a DNA construct represented as PsUrr-SS-FP-Xincluding a promoter (SEQ ID NO:56) operably linked to a syntheticspider silk protein sequence (SEQ ID NO:20), operably linked to afluorescent protein coding sequence (SEQ ID NO:13) which is operablylinked to a transcription terminator sequence.

Example 40—Exemplary Methods for Designing Synthetic Spider SilkProteins for Expression in Plants

The following methods for designing synthetic spider silk proteins arebased on the amino acid composition of spider silk proteins and howrepetitive regions of amino acid sequences contribute to thestructural/physical properties of spider silk proteins.

Synthetic spider silk proteins may be comprised of a series of tandemexact repeats of amino acid sequence regions identified as possessing aparticular spectrum of physical properties. Exact repeats compriseregions of amino acid sequences that are duplicated precisely.Alternatively, synthetic spider silk proteins may be comprised of aseries of tandem inexact repeats identified as having a spectrum ofphysical properties. Inexact repeats may comprise regions of amino acidsequences in which at least one amino acid in the basic inexact repeatunit has been altered, as long as the alteration does not change thespectrum of physical properties characteristic of the basic inexactrepeat unit.

In order to increase the tensile strength of a minor ampullate silk, forexample, to adapt it for applications in which strength and very littleelasticity are needed, such as bulletproof vests, the (GA)n regions maybe replaced by (A)n regions. This change would increase the tensilestrength. The typical MiSp1 protein has sixteen (GA) units. Replacingeight (GA) regions, for example, with (A) regions would increase thetensile strength from 100,000 psi to at least 400,000 psi. Moreover, ifthe (A)n regions are as long as the (GA)n regions the tensile strengthwould increase to greater than 600,000 psi.

To create a fiber with high tensile strength and greater elasticity thanmajor ampullate silk, the number of regions may be increased from 4-5regions, the range of regions typically found in naturally occurringmajor ampullate spider silk proteins, to a larger number of regions. Forexample, if the number are increased to 10-12 regions, the elasticitywould increase to 50-60%. If the number are further increased to 25-30regions, the elasticity would be near 100%. Such fibers may be used toadvantage in coverings for wounds (for example, burn wounds) tofacilitate easier placement and provide structural support. Such fibersmay also be used for clothing and as fibers in composite materials.

The tensile strength of a very elastic flagelliform silk may beincreased by replacing some of the units with (A)n regions. Aflagelliform silk protein contains an average of 50 units per repeat.Replacing two units in each repeat with (A) regions may, therefore,increase the tensile strength of a flagelliform silk by a factor of fourto achieve a tensile strength of about 400,000 psi. Uses for suchflagelliform silk proteins are similar to those described for majorampullate proteins having augmented elasticity. The flagelliformproteins have additional utility in that the spacer regions confer theability to attach functional molecules like antibiotics and/or growthfactors (or combinations thereof) to composites comprising flagelliformproteins.

Synthetic spider proteins may also comprise the following elasticsequence motifs: from Araneus dragline; from Lactrodectus dragline; andfrom Argiope dragline. Genes comprising 2, 4, 8 and 16 repeats of thesemotifs may be constructed. The naturally occurring linker, connected toa poly-alanine segment of eight residues may be used to flank eachrepeat unit. The poly-alanine segment may be used as in the naturalprotein for fiber formation. This entire unit may be increased up to 16repeat units to generate an encoded protein of 70-80 kD. Varying thenumber of these motifs alters the amount of elasticity from about 30%(for a synthetic spider silk protein coding sequence comprising tworepeats derived from Araneus) to nearly 200% (for a synthetic spidersilk protein coding sequence comprising sixteen repeats derived fromArgiope). Varying the sequence of the motifs modifies the elasticmodulus (higher with Araneus, lower with Argiope).

Genes encoding synthetic spider proteins derived from one of the AraneusMaSp2 protein analog genes may also be constructed. Such Araneus MaSp2protein analog genes comprise beta-sheet motifs from poly-alaninesegments of 5 and 14 residues that are the smallest and largestpoly-alanine tracts found in the major ampullate silk proteins. Thesesegments may also be constructed the novel sequence motif (gly-ala orgly-val) with the numerical value of n ranging from 3 to 8, the rangeobserved in natural spider silk proteins. Varying the length andsequence of the beta-sheet region alters the tensile strength fromapproximately that of the typical minor ampullate silk (100,000 psi) toat least 600,000 psi, double that of dragline silk. Moreover, thespecific sequence of the repeat influences the tensile strength of thefiber.

While a number of exemplary aspects and embodiments have been discussedabove, those of skill in the art will recognize certain modifications,permutations, additions and sub-combinations thereof. It is thereforeintended that the following appended claims and claims hereafterintroduced are interpreted to include all such modifications,permutations, additions, and sub-combinations as are within their truespirit and scope.

The present invention may be embodied in other specific forms withoutdeparting from its spirit or essential characteristics. The describedembodiments are to be considered in all respects only as illustrativeand not restrictive. The scope of the invention is, therefore, indicatedby the appended claims rather than by the foregoing description. Allchanges which come within the meaning and range of equivalency of theclaims are to be embraced within their scope.

The present invention, in various embodiments, include components,methods, processes, systems and/or apparatus substantially as depictedand described herein, including various embodiments, subcombinations,and subsets thereof. Those of skill in the art will understand how tomake and use the present invention after understanding the presentdisclosure.

The present invention, in various embodiments, includes providingdevices and processes in the absence of items not depicted and/ordescribed herein or in various embodiments hereof, including in theabsence of such items as may have been used in previous devices orprocesses (e.g., for improving performance, achieving ease and/orreducing cost of implementation).

The foregoing discussion of the invention has been presented forpurposes of illustration and description. The foregoing is not intendedto limit the invention to the form or forms disclosed herein. In theforegoing Detailed Description for example, various features of theinvention are grouped together in one or more embodiments for thepurpose of streamlining the disclosure. This method of disclosure is notto be interpreted as reflecting an intention that the claimed inventionrequires more features than are expressly recited in each claim. Rather,as the following claims reflect, inventive aspects lie in less than allfeatures of a single foregoing disclosed embodiment. Thus, the followingclaims are hereby incorporated into this Detailed Description, with eachclaim standing on its own as a separate preferred embodiment of theinvention.

Moreover, though the description of the invention has includeddescription of one or more embodiments and certain variations andmodifications, other variations and modifications are within the scopeof the invention (e.g., as may be within the skill and knowledge ofthose in the art, after understanding the present disclosure). It isintended to obtain rights which include alternative embodiments to theextent permitted, including alternate, interchangeable and/or equivalentstructures, functions, ranges or acts to those claimed, whether or notsuch alternate, interchangeable and/or equivalent structures, functions,ranges or acts are disclosed herein, and without intending to publiclydedicate any patentable subject matter.

The use of the terms “a,” “an,” and “the,” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. Forexample, if the range 10-15 is disclosed, then 11, 12, 13, and 14 arealso disclosed. All methods described herein can be performed in anysuitable order unless otherwise indicated herein or otherwise clearlycontradicted by context. The use of any and all examples, or exemplarylanguage (e.g., “such as”) provided herein, is intended merely to betterilluminate the invention and does not pose a limitation on the scope ofthe invention unless otherwise claimed. No language in the specificationshould be construed as indicating any non-claimed element as essentialto the practice of the invention.

We claim:
 1. An artificial, synthetic spider silk protein sequence,wherein said protein sequence is selected from the group consisting ofSEQ ID NO: 36, SEQ ID NO: 38 and SEQ ID NO:
 40. 2. An expression vectorcomprising a nucleic acid sequence encoding at least one of the spidersilk proteins of claim
 1. 3. A host cell comprising the expressionvector of claim
 2. 4. A silk fiber comprising at least one of theproteins of claim
 1. 5. A nucleic acid sequence encoding an artificialsynthetic spider silk protein, wherein said sequence is selected fromthe group consisting of SEQ ID NO: 35, SEQ ID NO: 37 and SEQ ID NO: 39.6. An expression vector comprising at least one of the nucleic acidsequences of claim
 5. 7. A host cell comprising the expression vector ofclaim
 6. 8. A silk fiber comprising at least one of the proteinsproduced from at least one of the nucleic acid sequences of claim 5.